Methods and compositions for detecting multiple analytes with a single signal

ABSTRACT

Compositions, methods, and devices for the detection of multiple analytes with a single signal are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/789,002, filed Mar. 7, 2013, which claims priority to U.S.Provisional Application No. 61/608,774, filed Mar. 9, 2012, each ofwhich is hereby incorporated by reference in its entirety.

FIELD OF INVENTION

Embodiments are directed to, in part, the detection of multiple analyteswith a single signal.

BACKGROUND OF INVENTION

The detection of multiple analytes often requires the use of multiplesignals or multiple reactions, spots, or wells to determine if a samplehas multiple analytes. This can complicate interpretation and, in caseswhere an adulterant is classified as having two or more detectablecharacteristics, can make identification challenging for the end-user.Thus, to simplify and provide a consolidated qualitative report to theend-user, there is a need for methods and compositions that enable thedetection of multiple analytes in a sample with a single signal. Thepresent invention satisfies this need and others.

SUMMARY OF THE INVENTION

The present invention provides methods of concurrently detecting a firstanalyte and a second analyte comprising: contacting a solid support witha first analyte, a second analyte, a bridge unit comprising a secondcapture reagent, and a signal detection unit comprising a third capturereagent; and detecting the presence or absence of the signal detectionunit which indicates the presence or absence of the first analyte andsecond analyte concurrently, wherein a first capture reagent is affixedto the solid support; the first analyte comprises a first interactionunit that binds to the first capture reagent and a second interactionunit that binds to the bridge unit; and the second analyte comprises afirst interaction unit that binds the bridge unit and a secondinteraction unit that binds to the signal detection unit.

The present invention also provides methods of concurrently detecting afirst analyte, a second analyte, and a third analyte with a singlesignal comprising: contacting the first, second, and third analytes witha solid support, a first bridge unit, a second bridge unit, and a signaldetection unit; and detecting the presence of the signal detection unitwhich indicates the presence of the first, second, and third analytesconcurrently with a single signal, wherein: the first analyte comprisesa first interaction unit and a second interaction unit; the secondanalyte comprises a first interaction unit and a second interactionunit; the third analyte comprises a first interaction unit and a secondinteraction unit; the solid support comprises a first capture reagentthat binds to the first interaction unit of the first analyte; the firstbridge unit binds to the second interaction unit of the first analyteand the first interaction unit of the second analyte; the second bridgeunit binds to the second interaction unit of the second analyte and thefirst interaction unit of the third analyte; and the signal detectionunit binds to the second interaction unit of the third analyte. Theinteraction units can be different from one another on each of theanalytes.

In some embodiments, methods of concurrently detecting a first analyteand a second analyte are provided, the method comprising: contacting asolid support with a first analyte of interest, a second analyte ofinterest, a bridge unit comprising a second capture reagent, and asignal detection unit comprising a third capture reagent; and detectingthe presence or absence of the signal detection unit which indicates thepresence or absence of the first analyte of interest and second analyteof interest concurrently, wherein: a first capture reagent is affixed tothe solid support; the first analyte of interest comprises a firstinteraction unit that binds to the first capture reagent and a secondinteraction unit that binds to the bridge unit; and the second analyteof interest comprises a first interaction unit that binds the bridgeunit; a signal detection unit that binds to the second analyte, to thesecond analyte's first interaction unit or a second interaction unit, toa component of the first and second analyte complex or bridge unit thatthat is only present when the complex contains the first and secondanalyte.

Embodiments described herein also provide complexes comprising a solidsupport, a first analyte, a second analyte, a bridge unit, and a signaldetection unit wherein each member of the complex binds to each otherdirectly or indirectly.

Embodiments described herein also provide complexes comprising a solidsupport, a first analyte, a second analyte, a third analyte, a firstbridge unit, a second bridge unit, and a signal detection unit, whereinthe solid support, first analyte, second analyte, third analyte, firstbridge unit, second bridge unit, and signal detection unit are bound toeach other directly or indirectly.

Methods of concurrently detecting a first analyte of interest and asecond analyte of interest are provided herein. In some embodiments, themethod comprises contacting a solid support with a first analyte ofinterest, a second analyte of interest, a bridge unit comprising asecond capture reagent, and a signal detection unit comprising a thirdcapture reagent; and detecting the presence or absence of the signaldetection unit which indicates the presence or absence of the firstanalyte of interest and second analyte of interest concurrently, whereina first capture reagent is affixed to the solid support; the firstanalyte of interest comprises a first interaction unit that binds to thefirst capture reagent and a second interaction unit that binds to thebridge unit; and the second analyte of interest comprises a firstinteraction unit and a second interaction unit, wherein the firstinteraction unit binds the bridge unit; a signal detection unit thatbinds to: i) the second analyte, ii) to the second analyte's firstinteraction unit or second interaction unit, iii) to a component of thefirst and second analyte complex, or iv) a component of ananalyte-bridge complex that is only present when the complex containsthe first and second analytes.

In some embodiments, the first and second interaction unit of the firstanalyte of interest and the first and second interaction unit of thesecond analyte of interest are each, independently, a heterologousinteraction unit. In some embodiments, the second interaction unit ofthe first analyte of interest and the first interaction unit of thesecond analyte of interest comprise the same heterologous interactionunit. In some embodiments, the second interaction unit of the firstanalyte of interest and the first interaction unit of the second analyteof interest comprise different heterologous interaction units. In someembodiments, the first interaction unit of the first analyte of interestand the second interaction unit of the second analyte of interestcomprise the same heterologous interaction unit. In some embodiments,the first interaction unit of the first analyte of interest and thesecond interaction unit of the second analyte of interest comprisedifferent heterologous interaction units.

Methods of concurrently detecting a first analyte of interest, a secondanalyte of interest, and a third analyte of interest with a singlesignal are provided. In some embodiments, the method comprisescontacting the first, second, and third analytes of interest with asolid support, a first bridge unit, a second bridge unit, and a signaldetection unit; and detecting the presence of the signal detection unitwhich indicates the presence of the first, second, and third analytes ofinterest concurrently with a single signal, wherein: the first analyteof interest comprises a first interaction unit and a second interactionunit; the second analyte of interest comprises a first interaction unitand a second interaction unit; the third analyte of interest comprises afirst interaction unit and a fifth interaction unit; the solid supportcomprises a first capture reagent that binds to the first interactionunit of the first analyte of interest; the first bridge unit binds tothe second interaction unit of the first analyte of interest and thefirst interaction unit of the second analyte of interest; the secondbridge unit binds to the second interaction unit of the second analyteof interest and the first interaction unit of the third analyte ofinterest; and the signal detection unit binds to: i) the third analyte,ii) to the third analyte's first interaction unit or second interactionunit, iii) to a component of the first, second, or third analytecomplex, or iv) a component of an analyte-bridge complex that is onlypresent when the complex contains the first, second, and third analytes.

In some embodiment, the bridge units described herein are multivalentcapture reagents. In some embodiments, the multivalent capture reagentis an immunoglobulin. In some embodiments, the immunoglobulin is IgM.The bridge unit can also be biotin.

Methods of concurrently detecting a plurality of analytes with a singlesignal with a device are provided. In some embodiments, the methodcomprises a) contacting a device for detecting a plurality of analyteswith a single signal with one or more samples comprising a plurality ofanalytes, wherein the device comprises: a housing comprising: an inletopening in fluid contact with a conjugate pad; a force member; aslidable locking member contacting the force member; an attachmentmember contacting the force member; a sliding button contacting theattachment member; and a detection membrane system comprising theconjugate pad, a test membrane, and an absorbent member, at least aportion of the conjugate pad, test membrane, and absorbent member aresubstantially parallel to each other, the force member contacts thedetection membrane system and is capable of applying pressuresubstantially perpendicular to the detection membrane system, thesliding button moves the slidable locking member, the conjugate padcomprises a signal detection unit comprising a third capture reagent;the test membrane comprises a first capture reagent affixed to the testmembrane; wherein the one or more samples comprises a first analyte ofinterest, a second analyte of interest, and a bridge unit comprising asecond capture reagent, wherein the first analyte of interest comprisesa first interaction unit that binds to the first capture reagent and asecond interaction unit that binds to the bridge unit, and the secondanalyte of interest comprises a first interaction unit that binds thebridge unit and a second interaction unit; wherein the signal detectionunit comprising the third capture reagent binds to: i) the secondanalyte, ii) to the second analyte's first interaction unit or secondinteraction unit, iii) to a component of the first and second analytecomplex, or iv) a component of an analyte-bridge complex that is onlypresent when the complex contains the first and second analytes; and b)detecting the presence or absence of the signal detection unit whichindicates the presence or absence of the first analyte of interest andsecond analyte of interest concurrently.

In some embodiments, the method comprises moving the conjugate pad aftera portion of the one or more samples has contacted and flowed throughthe conjugate pad, thereby exposing at least a portion of the testmembrane for detection of the signal detection unit to indicate thepresence or absence of the plurality of analytes with a single signal.In some embodiments, the conjugate pad is moved by moving the slidablelocking member. In some embodiments, the first and second analyte areamplicons. In some embodiments, the first and second analytes are PCRreaction products. In some embodiments, the first analyte's firstinteraction unit is a digoxigenin label. In some embodiments, the firstanalyte's second interaction unit is a rhodamine label. In someembodiments, the second analyte's first interaction unit is a rhodaminelabel. In some embodiments, the second analyte's second interaction unitis a fluorescein label. In some embodiments, the third capture reagentbinds to the second analyte's second interaction unit. In someembodiments, the third capture reagent is a biotinylated capturereagent. In some embodiments, the signal interaction unit is coated withstreptavidin. In some embodiments, the signal interaction unit isstreptavidin coated colloidal gold. In some embodiments, the first andsecond analytes are nucleic acid amplification products, wherein: thefirst analyte comprises a digoxigenin label and a rhodamine label; thesecond analyte comprises a rhodamine label and a fluorescein label; thefirst capture reagent is an anti-digoxigenin label antibody; the secondcapture reagent is an anti-rhodamine label antibody; the third capturereagent is a biotinylated anti-fluorescein label antibody; and thesignal interaction unit is streptavidin coated colloidal gold.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates, among other aspects, the representative detection oftwo analytes with a single signal.

FIG. 2 illustrates, among other aspects, the representative detection ofthree analytes with a single signal.

FIG. 3 illustrates, among other aspects, two amplification productsbeing detected with colloidal gold.

FIG. 4 illustrates, among other aspects, a multi-component bridging unit

FIG. 5 illustrates, among other aspects, the representative detection oftwo analytes with a single signal using a multi-component bridging unit.

FIG. 6 illustrates, among other aspects, the signal detection unitbinding to a component of the bridging unit that is only present whenthe plurality of analytes is present in the complex.

FIG. 7 illustrates, among other aspects, a non-limiting workflow fordetecting a plurality of analytes with a single signal.

FIG. 8 depicts a perspective view of a representative device accordingto some embodiments of the present invention.

FIG. 9 depicts some components of a representative device according tosome embodiments of the present invention.

FIG. 10 depicts some components of a representative device according tosome embodiments of the present invention.

FIG. 11 depicts some components of a representative device according tosome embodiments of the present invention.

FIG. 12 depicts some components of a representative device in variouspositions according to some embodiments of the present invention.

FIG. 13: Depicts a lateral view of some components of a representativedevice according to some embodiments of the present invention.

FIG. 14 depicts a lateral view of some components of a representativedevice according to some embodiments of the present invention.

FIG. 15A depicts a lateral view of some components of a representativedevice according to some embodiments of the present invention.

FIG. 15B depicts a view of some components, such as but not limited to,a non-flexible attachment member, of a representative device accordingto some embodiments of the present invention.

FIG. 15C depicts a perspective view of a representative device accordingto some embodiments of the present invention.

FIG. 15D depicts a perspective view of a representative device accordingto some embodiments of the present invention.

FIG. 16 depicts a flexible attachment member attached to a conjugatepad.

FIG. 17 depicts membranes in a representative housing member.

FIG. 18 depicts a side view and a top view of a representative deviceaccording to some embodiments of the present invention.

FIG. 19 depicts one type of analyte detection membrane system for arepresentative device according to some embodiments of the presentinvention.

FIG. 20 depicts one type of analyte detection membrane system for arepresentative device according to some embodiments of the presentinvention.

FIG. 21 depicts one type of analyte detection membrane system for arepresentative device according to some embodiments of the presentinvention.

FIG. 22 depicts one type of analyte detection membrane system for arepresentative device according to some embodiments of the presentinvention.

FIG. 23 depicts representative force members for a representative deviceaccording to some embodiments of the present invention.

FIGS. 24A-24D depict a representative device according to someembodiments of the present invention.

FIGS. 25A-25C depict a representative device according to someembodiments of the present invention.

FIG. 26 depicts representative devices according to some embodiments ofthe present invention.

FIGS. 27A-27B depict a view of a representative device according to someembodiments of the present invention.

FIG. 28 depicts an underneath view of a representative device accordingto some embodiments of the present invention.

FIG. 29 depicts an exploded view of a representative device according tosome embodiments of the present invention.

FIG. 30 depicts an interior view of a representative device according tosome embodiments of the present invention.

FIGS. 31A-31B depict a cross-sectional view of a representative deviceaccording to some embodiments of the present invention.

FIG. 32 depicts an exploded view of a representative device according tosome embodiments of the present invention.

FIG. 33 depicts an interior view of a representative device according tosome embodiments of the present invention.

FIG. 34 depicts a cross-sectional view of a representative deviceaccording to some embodiments of the present invention.

FIG. 35 depicts a representative movable locking member according tosome embodiments of the present invention.

FIG. 36 depicts a representative housing according to some embodimentsof the present invention.

FIG. 37 depicts a representative housing according to some embodimentsof the present invention.

FIG. 38A depicts a representative device according to some embodimentsof the present invention.

FIG. 38B depicts a representative device according to some embodimentsof the present invention.

FIG. 39 depicts an enlarged view of a representative device according tosome embodiments of the present invention.

FIG. 40 depicts an exploded view of a cartridge and analyte detectionmembrane system according to some embodiments of the present invention.

FIG. 41 depicts a representative device according to some embodiments ofthe present invention.

FIG. 42 depicts a representative device according to some embodiments ofthe present invention.

FIGS. 43A-43C depict a representative device according to someembodiments of the present invention.

FIG. 44 depicts an exploded view of a representative device according tosome embodiments of the present invention.

FIG. 45 depicts an exploded view of a representative device according tosome embodiments of the present invention.

DESCRIPTION OF EMBODIMENTS

Before compositions and methods provided herein are described, it is tobe understood that the embodiments are not limited to the particularprocesses, compositions, or methodologies described, as these may vary.It is also to be understood that the terminology used in the descriptionis for the purpose of describing some embodiments, and is not intendedto limit the scope of the embodiments.

Various methods and embodiments are described herein. The methods andembodiments can be combined with one another. The definitions andembodiments described herein are not limited to a particular method orexample unless the context clearly indicates that it should be solimited.

As used herein, the phrase “detection of an analyte,” “detecting ananalyte” refers the detection of multiple analytes with a single signal.The detection of multiple analytes can be, as described herein, atleast, or exactly, 2, 3, 4, or 5 analytes with a single signal.

It must be noted that, as used herein and in the appended claims, thesingular forms “a”, “an” and “the” include plural reference unless thecontext clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art. Although anymethods similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the present invention, thepreferred methods are now described. All publications mentioned hereinare incorporated by reference in their entirety to the extent to supportthe presently described subject matter. Nothing herein is to beconstrued as an admission that the subject matter is not entitled toantedate such disclosure by virtue of prior invention.

As used herein, the term “about” means plus or minus 10% of thenumerical value of the number with which it is being used. Therefore,about 50% means in the range of 45%-55%. Additionally, in phrase “aboutX to Y,” is the same as “about X to about Y,” that is the term “about”modifies both “X” and “Y.”

As used herein, the term “optional” or “optionally” means that thesubsequently described structure, event or circumstance may or may notoccur, and that the description includes instances where the eventoccurs and instances where it does not.

As used herein, the term “sample” means any fluid medium or liquid thatmay contains a particular item (e.g. analyte) or suspected of containinga particular item. In some embodiments, samples may be used which arehigh in dissolved solids without further processing, and samplescontaining high solids (non-dissolved) may be analyzed, in someembodiments, through the use of a filter or used in conjunction withadditional manual steps. Samples may also be non-filtered or purifiedprior to being used in a method or device described herein. Samples maybe a liquid, a suspension, extracted or dissolved sample, or asupercritical fluid. If a sample is going to be used in a flow device(vertical or lateral) some flow properties must exist in the sample orextract to allow flow through the devices and systems described herein.Examples of samples include, but are not limited to, blood, food swabs,food extracts, food suspensions, food cultures, bacterial cultures,viral cultures, amplification reactions, saliva, biological fluid, PCRreactions, and the like. The sample can also be derived from a anothersample. For example, a PCR reaction can be performed on a nucleic acidmixture that has been extracted, isolated, and/or purified from anothersample (e.g., food, cellular, viral, bacterial, blood, and the like).The PCR reaction would be considered to be a sample derived from anothersample.

A “food suspension” refers to raw or cooked food that has been placed orsuspended in a solution. The food solution may be mixed, vortexed orblended. A “food culture” is a food sample that is cultured underconditions to enrich the sample. This process can also be referred to as“enrichment.” The enrichment can be used to facilitate sample analysisto better detect the presence or absence of multiple analytes with asingle signal. The sample can also be a reaction sample that is derivedfrom a different sample. An example of a reaction sample is an“enrichment.” For example, a blood or food sample may be processed (e.g.cultured, purified, separated into components, and the like) and theprocessed sample can be tested for the detection of multiple analytes.In some embodiments, two analytes are detected in a blood sample or afood sample. In some embodiments, the analytes can be detected byperforming two amplification reactions that are specific for the twoanalytes and then the two amplification products can be detected with asingle signal to detect the presence of the two analytes in a sampleconcurrently. In some embodiments, three analytes are detected using asingle signal. The detection can be concurrent, that is the signal isonly generated when all the analytes are present in the same sample. Theconcurrent signal generation can be effectuated through the creation ofa bridging complex, which is described herein. Non-limiting embodimentsof the bridging complex can be seen in FIGS. 1-3.

As used herein, the term “solid support” means a material that issubstantially insoluble in a selected system, or which can be readilyseparated (e.g., by precipitation) from a selected system in which it ispresent. Solid supports useful in practicing the present methods caninclude groups that are activated or capable of activation to allowcertain compounds or molecules (e.g. capture reagents, antibodies, andthe like) to be bound to the solid support. The solid support may, forexample, be agarose, sepharose, polyacrylamide, agarose/polyacrylamideco-polymers, dextran, cellulose, polypropylene, polycarbonate,nitrocellulose, glass paper, or any other suitable substance capable ofproviding a suitable solid support. In some embodiments, the solidsupport may be in the form of granules, a powder or a gel suitable foruse in chromatography. The solid support can also be a membrane, such anitrocellulose, PVC, and the like. Other types of membranes can also beused and there is no specific requirement for the type of membrane thatcan be used. In some embodiments, the solid support is a test membrane.Examples of test membranes are described herein.

As used herein, the term “analyte” includes, but is not limited to,antigens, nucleic acid molecules encoded by a cell, virus, bacteria orother type of microorganism, amplification products (e.g. amplicons), apeptide, a sugar, and the like. In some embodiments, the analyte is notan antibody or functional fragment thereof. Nucleic acid molecules canbe detected as described herein by using the methods described herein incombination with other known methods or devices, such as amplificationmethods (e.g. PCR, RT-PCR, and the like), hybridization methods, labeledprimers, and the like. The term “target molecule” can be usedinterchangeably with the term “analyte.” The amplification methods canbe used to amplify the amount of nucleic acid molecules present in asample to facilitate the detection of the analyte. Other types ofanalytes that can be detected using the methods described hereininclude, but are not limited to antigens, antibodies, receptors,ligands, chelates, proteins, enzymes, nucleic acids, DNA, RNA,pesticides, herbicides, inorganic or organic compounds, or any materialfor which a specific binding reagent may be found. The analyte can alsorefer to different epitopes present on the same protein or polypeptide.The analyte can also refer to analytes from pathogenic or non-pathogenicorganisms. The analytes can also be referred to as an analyte ofinterest in a sample. That is, the analyte can be referred to as anagent that a user is determining the presence or absence of in a sample.

As discussed herein, the analyte can be an amplification product, suchas a product of a PCR reaction. The PCR product is amplifying a nucleicacid sequence from a test sample. Thus, detection of the PCR product insample is determining whether the nucleic acid sequence that the PCRproduct is based upon is present in the initial sample. For example, ifone of skill in the art is determining whether a food sample iscontaminated with E. Coli, nucleic acid sequences that are specific forE. Coli can be amplified (e.g. by PCR) and then detected according tothe methods described herein. The detection of the amplificationproducts (i.e. amplicons) indicates that the food sample contained thenative nucleic acid sequences that are specific for E. Coli. This isexample is non-limiting and can be applied to detecting other nucleicacid sequence or other types of analytes present in a native sample. Theanalyte can be what is in the initial sample or an analyte that isderived from the initial sample by, for example, using PCR. When theplurality of analytes is being detected with a single signal accordingto the methods provided herein, the analytes can also have heterologoustags or interaction units, and the modified analyte is also referred toas the analyte. In some embodiments, the analyte will be free ofheterologous interaction units, such as fluorescent tags, biotin,digoxigenin, and the like.

An analyte is different from a reagent that is used to detect thepresence or absence of an analyte. Thus, a reagent that is added to thesample to determine if the analyte is present is not an analyte ofinterest. For example, in a typical sandwich assay, a first antibody isattached to a solid support. The solid support coated with an antibodyis contacted with a sample to determine the presence or absence of anantigen that binds to the antibody. A secondary antibody is then alsoadded to detect the antigen. The presence of the secondary antibody isthen often detected by the addition of a third antibody that has, forexample. An enzyme conjugated to it so that it can be detected throughvarious means (e.g. HRP-linked antibodies). The secondary antibody isnot an analyte of interest because it is a reagent used to detect theprimary antigen. Therefore, a sandwich assay does not detect thepresence of a plurality of analytes with a single signal according tothe methods described herein because the secondary antibody is areagent, or tool, to detect the presence or absence of the antigen, oran analyte of interest. An analyte is also not a component or portionthat is found on a bridging entity. For example, in U.S. PublishedApplication No. 2010/0273145 FIGS. 1 and 2 show an analyte binding to abridging entity, which then binds to a signaling entity to detect thepresence of the analyte. Neither the bridging unit, or any portionthereof, or the signaling entity is an analyte or analyte of interest.These components are reagents used to detect the analyte, which in thecase of U.S. Published Application No. 2010/0273145 is the detection ofa single analyte. U.S. Published Application No. 2010/0273145 is herebyincorporated by reference with regards to its figures and theexplanation of their components.

In some embodiments, the analyte is a protein, such as a pathogenprotein. A pathogen protein refers to a protein that is from a pathogen.Examples of pathogens include, but are not limited to, viruses,prokaryotes and, for example, pathogenic eukaryotic organisms such asunicellular pathogenic organisms and multicellular parasites. Pathogensalso can include protozoan pathogens which include a stage in the lifecycle where they are intracellular pathogens. As used herein, the term“intracellular pathogen” means a virus or pathogenic organism that, atleast part of its reproductive or life cycle, exists within a host celland therein produces or causes to be produced, pathogen proteins. Apathogen can also be a food-borne pathogen.

Bacterial pathogens include, but are not limited to, such as bacterialpathogenic gram-positive cocci, which include but are not limited to:pneumococcal, staphylococcal, and streptococcal. Pathogenicgram-negative cocci include, but are not limited to: meningococcal andgonococcal. Pathogenic enteric gram-negative bacilli include, but arenot limited to: enterobacteriaceae, pseudomonas, acinetobacteria,eikenella, melioidosis, salmonella, shigellosis, hemophilus, chancroid,brucellosis, tularemia, yersinia (pasteurella), streptobacillusmoniliformis, spirilum, Listeria monocytogenes, Erysipelothrixrhusiopathiae, diphtheria, cholera, anthrax, donovanosis (granulomainguinale), and bartonellosis. Pathogenic anaerobic bacteria include,but are not limited to, those that are responsible for: tetanus,botulism, other clostridia, tuberculosis, leprosy, and othermycobacteria. Pathogenic spirochetal diseases include, but are notlimited to: syphilis, treponematoses, yaws, pinta and endemic syphilis,and leptospirosis. Other infections caused by higher pathogen bacteriaand pathogenic fungi include, but are not limited to: actinomycosis,nocardiosis, cryptococcosis, blastomycosis, histoplasmosis, andcoccidioidomycosis, candidiasis, aspergillosis, mucormycosis,sporotrichosis, paracoccidiodomycosis, petriellidiosis, torulopsosis,mycetoma, chromomycosis, and dermatophytosis. Rickettsial infectionsinclude, but are not limited to, rickettsia and rickettsioses. Examplesof mycoplasma and chlamydial infections include, but are not limited to:mycoplasma pneumonia, lymphogranuloma venereum, psittacosis, andperinatal chlamydial infections. Pathogenic protozoans and helminths andinfectious eukaryotes thereby include, but are not limited to:amebiasis, malaria, leishmaniasis, trypanosomiasis, toxoplasmosis,pneumocystis carinii, babesiosis giardiasis trichinosis filariasisschistosomiasis, nematodes, trematodes or flukes, and cestode (tapeworm)infections. Bacteria also include, but are not limited to, Listeria, E.coli, Campylobacter species, and Salmonella species. In someembodiments, E. coli is E. coli 0157.

Examples of viruses include, but are not limited to, HIV, Hepatitis A,B, and C, FIV, lentiviruses, pestiviruses, West Nile Virus, measles,smallpox, cowpox, ebola, coronavirus, and the like. Other pathogens arealso disclosed in U.S. Patent Application Publication No. 20080139494,which are incorporated herein by reference.

In some embodiments, the pathogen is a food borne pathogen. The analytecan be present on a food borne pathogen. Food borne pathogens arepathogens (e.g. viral or bacterial) that cause illness after eatingcontaminated food. The food itself does not directly cause the illness,but it is rather the consumption of the food borne pathogen that ispresent on the food that causes the illness. In some embodiments, thefood borne pathogen is E. coli, Listeria, a Campylobacter species, or aSalmonella species. In some embodiments, the analyte is chosen from afood borne pathogen analyte. For example, the food borne pathogenanalyte can be, but is not limited to, chosen from an E. coli analyte, aListeria analyte, a Campylobacter species analyte, or a Salmonellaspecies analyte. In some embodiments, the analyte is the specificO-Antigen. In some embodiments, the O-antigen is the E. coli antigenand/or a Salmonella species O-antigen and can be used for E. coli andSalmonella detection. In some embodiments, the analyte is a flagellinantigen. In some embodiments, the analyte is the Campylobacter flagellinantigen. In some embodiments the analyte is a virulence factor gene suchas the Shiga toxin gene amplified from pathogenic E. coli or Salmonella.In some embodiments, the analyte is a DNA or RNA sequence that isamplified via an amplification method (e.g. PCR or RT-PCR) and thendetected according to the methods described herein.

As described herein, an analyte can be an amplification product. Theamplification product, such as PCR product (e.g. a double stranded PCRproduct), can be labeled with interaction units. The production of alabeled amplification product with the units can be made by the use ofprimers labeled or conjugated with the two interaction units. In someembodiments, an analyte will have two different interaction units sothat the bridging complex can be assembled and the detection of multipleanalytes is possible through a signal detection unit.

As used herein, the term “signal detection unit” means a unit that canbe detected to determine if the analyte or analytes are present in asample. The signal detection unit can be any reagent or composition thatcan be detected. In some embodiments, the signal detection unit isattached to a capture reagent. Thus, the signal detection unit can beused to detect the presence of the capture reagent binding to itsspecific binding partner. The capture reagent can comprise a detectionreagent directly or the capture reagent can further comprise a particlethat comprises the detection reagent. In some embodiments, the capturereagent and/or particle comprises a color, colloidal gold, a radioactivetag, a fluorescent tag, or a chemiluminescent substrate. In someembodiments, the signal detection unit comprises a near-infrared orinfrared tag or substrate. In some embodiments, the signal detectionunit comprises a color, colloidal gold, a radioactive tag, a fluorescenttag, or a chemiluminescent substrate. In some embodiments, the signaldetection unit comprises a nanocrystal, functionalized nanoparticles,up-converting nanoparticles, cadmium selenide/cadmium sulfide fusionnanoparticles, quantum dots, and a Near-Infrared (NIR) fluorophore ormaterial (such as, but not limited to, materials such as lanthanideclusters and phthalocyanines, as well as light emitting-diodesconsisting of CuPc, PdPc, and PtPc) capable of emitting light in the NIRspectrum. In some embodiments, a capture reagent and/or particle isconjugated to the signal detection unit, such as but not limited to,colloidal gold, silver, radioactive tag, fluorescent tag, or achemiluminescent substrate, near-infrared compound (e.g. substrate,molecule, particle), or infrared compound (e.g. substrate, molecule,particle), nanoparticle, emissive nanoparticle, quantum dot, magneticparticle, or an enzyme.

The signal detection unit can also be, for example, a viral particle, alatex particle, a lipid particle, a fluorescent particle, anear-infrared particle, or infrared particle. As used herein, the term“fluorescent particle” means a particle that emits light in thefluorescent spectrum. As used herein, the term “near-infrared particle”means a particle that emits light in the near-infrared spectrum. As usedherein, the term “infrared particle” means a particle that emits lightin the infrared spectrum. In some embodiments, the colloidal gold has adiameter size of: about 20 nm, about 30 nm, or about 40 nm, or in therange of about 20-30 nm, about 20-40 nm, about 30-40 nm, or about 35-40nm. In some embodiments, the particle comprises a metal alloy particle.In some embodiments, the metal alloy particle has a diameter from about10 to about 200 nm. Examples of metal alloy particles include, but arenot limited to, gold metal alloy particles, gold-silver bimetallicparticles, silver metal alloy particles, copper alloy particles,Cadmium-Selenium particles, palladium alloy particles, platinum alloyparticles, and lead nanoparticles.

As discussed herein the signal detection can will bind to one of theanalytes. A non-limiting example of the signal detection unit binding toan analyte is shown in FIG. 1. FIG. 1, which is described in more detailherein, shows the signal detection unit 60 binding to the analyte 40through a capture reagent 50. However, the signal detection unit canalso bind to other portions of the complex. Any component that isnecessarily present only when both the plurality of analytes is presentin the complex can be a binding partner for the signal detection unit.Often, but not exclusively this will be one of the analytes, but canalso be a capture reagent that is bound to the analyte. In contrast, insome embodiments, the signal detection unit does not bind solely to theanalyte that is bound to the solid support, the solid support, or thecapture reagent bound directly to the solid support, if present on thesolid support. For example, in FIG. 1, the signal detection unit willnot bind directly to the solid support 10, the capture reagent 15, orthe analyte 20. Without being bound to any particular theory, if thesignal detection unit binds directly with the solid support 10, thecapture reagent 15, or the analyte 20, the method would provide a falsepositive as the signal would be detected without the plurality ofanalytes necessarily being present. For example, FIG. 6, illustrates thesignal detection unit binding to a component of a multi-componentbridging unit. Embodiments of the bridging unit, and a multi-componentbridging unit, are described herein and, for example, with references toFIGS. 4 and 5. FIG. 6 illustrates a signal detection unit 60 with itscapture reagent 50 binding to a component of the bridging unit 30. Thebridging unit comprises 30 a particle 34, a first capture reagent 31, asecond capture reagent 32, and a third capture reagent 33. FIG. 6illustrates the signal detection unit binding to the second capturereagent 32. The capture reagent 32 will only be present in the complexif both analytes are present in the complex. If capture reagent 32 isnot present this means that there is no bridged complex of the pluralityof analytes. Therefore, the signal detection unit will only be part ofthe complex if the plurality of analytes are present in the complex,thus avoiding false positives. If both analytes are not present thecapture reagent 32 will not be part of the complex, and, therefore,there will be no binding partner for the signal detection unit.Accordingly, the signal detection unit will only be detectable when theplurality of analytes are present. Therefore, in some embodiments, thesignal detection unit binds to any component that is only present whenthe plurality of analytes are also present. Other properties,characteristics, and structural features of the multi-component bridgeunit are also disclosed herein and are readily apparent based upon thepresent disclosure.

Examples of devices in which the presently described methods can be usedare described in, for example, in U.S. Pat. No. 8,012,770, U.S. patentapplication Ser. No. 13/360,528, filed Jan. 27, 2012, PCT PublicationNo. WO 2011/044574, each of which is incorporated herein by reference inits entirety. The presently describes methods, however, can be used withany number of devices or formats, such as multi-well plates, arrays,microarrays, or in an “ELISA” type format. Examples of devices are alsodescribed herein, but these examples are non-limiting. The methodsdescribed herein can also be used in conjunction with lateral flowdevices. In a lateral flow device the different portions of the deviceare in the same plane as opposed to a vertical flow device. Non-limitingexamples of the lateral flow devices can be found in U.S. Pat. Nos.6,485,982, 6,818,455, 6,951,631, 7,109,042, RE39,664, and the like, eachof which are hereby incorporated by reference. The lateral flow devicescan be adapted for the methods described herein as they are describedfor the vertical flow devices. In a lateral flow device, the region thatindicates a positive or negative result can comprise the capture reagentthat binds to one of the analytes. The bridge unit can either be presentin one of the lateral flow regions or mixed with the analytes beforeaddition to the device—this can also be done for other devices and solidsupports. The signal detection unit can also be incorporated into one ofthe lateral flow regions. As is clear from the present disclosure thetype of device or solid support is not critical and the methods can beadapted based upon the examples and embodiments described herein.

As used herein, the term “amplicon” means an amplification product suchas a nucleic acid molecule that is amplified by a PCR reaction or otheramplification reaction or method. As discussed herein, an amplicon canbe an analyte. The amplicon can be a double-stranded nucleic acidmolecule. The amplification product can be detected directly orindirectly through the use of antibodies or other capture reagentsystems, including those that are described herein. The amplificationproduct can also be detected through hybridization methods in whole orin part as described herein. The amplification product can also beproduced, for example, through RT-PCR or linear amplification.

In some embodiments, the amplicon is a PCR product. The PCR reactionproducts (e.g. amplicons) can be labeled such that they are detectableeither by another antibody or antibody like system, such as but notlimited to biotin-avidin/streptavidin system, systems, hapten systems,BRDU labeling of DNA, intercalating agents that label DNA, labeleddNTPS, and the like can also be used where the PCR products are labeled.The analyte, which can, for example, but not limited to, be a nucleicacid (single stranded or double stranded) and can be recognized ordetected with an antibody or other capture reagent system, such as thosedescribed herein. The nucleic acid molecule can be labeled with a biotinlabel or other type of label that can be detected using a methoddescribed herein. Other examples of labels include fluorescent labels.The fluorescent labels can be for example, fluorescein (e.g. fluoresceinisothiocyanate (FITC)), rhodamine (e.g. tetramethylrhodamine (TAMRA)),and the like. The amplicons can be generated with these labels by usinglabeled primers. The labels can be incorporated into the ampliconthrough the amplification procedure and, thus, become part of theanalyte. The labels would be considered heterologous tags because thelabels are not found in the native sequence that is used as the templatefor the amplicon. Capture reagents (e.g. antibodies) can be used thatbind to the labels to help in forming the complexes that are describedherein, which enable the detection of multiple analytes with a singlesignal. These labels can act as interaction units. A non-limitingexample of how the labels can act as interaction units such thatmultiple analytes can be detected with a single signal is shown in FIG.3.

For example, in some embodiments, a PCR reaction is performed with ahapten and/or biotin labeled DNA or RNA primers with homology to ananalyte nucleic acid sequence. The analyte nucleic acid sequence can be,but not limited to, a toxin gene and/or a toxin molecule (e.g. Shigatoxin) from a meat sample. The sample, however, can be any sample, andthe analyte can be any other type of analyte described herein. The PCRreactions can be performed to produce multiple analytes with theinteraction units. Following amplification with the primers, the PCRsample can be detected using a method described herein. The PCR reactioncan also be performed with digoxigenin and/or TAMRA and/or with FITC andTAMRA labeled primers. These can create the differentially labeledamplicons that can be bridged together through the use of capturereagents to enable the detection of multiple analytes with a singlesignal. An example of such a complex is shown in FIG. 3.

FIG. 3 illustrates a test membrane (i.e., solid support 10) with anAnti-Dig antibody (i.e., capture reagent 15), a Digoxigenin/TAMRAlabeled amplicon (i.e., a first analyte 20, a first interaction unit 21,and a second interaction unit 22), an anti-rhodamine antibody ((i.e.bridge unit 30), a FITC/TAMRA labeled amplicon (i.e., a second analyte40, a first interaction unit 41, and a second interaction unit 42); anda streptavidin-gold complex binding to a biotinylated anti-FITC antibody(i.e., a signal generation unit 60 and a third capture reagent 50).

Briefly, after the PCR reactions are performed, the amplicons can becontacted with a solid support, a bridging unit, and a signal detectionunit. The solid support can have a capture reagent that binds to aninteraction unit on the first analyte. The bridging unit can have, orbe, a capture reagent that binds to interaction units on the first andsecond analytes such that the binding to the interaction units on thefirst and second analytes brings the analytes together into a complex.The signal detection unit can bind to an interaction unit present on theone of the second analyte. The signal detection unit can then emit adetectable signal or the signal detection unit can be detected by theaddition of another detection system. For example, in FIG. 3, the signaldetection unit is a capture reagent (e.g. antibody) that binds to theinteraction unit on the second analyte. The signal detection unit isbiotinylated. The presence of the signal detection unit can be then bedetermined by the addition of streptavidin. The streptavidin will onlybind to a complex that has both analytes present. In the non-limitingexample shown in FIG. 3, the streptavidin is labeled with colloidal goldwhich enables the detection. However, other labels or detection systemscould be used to detect the streptavidin. In the embodiments of thevertical flow devices described herein, the test membrane is the solidsupport with the capture reagent, and the conjugate pad can comprise thesignal detection unit or the molecule that detects the binding of thesignal detection unit to the interaction unit of the second analyte.

FIG. 7 illustrates a non-limiting work flow procedure that could be usedto detect a plurality of analytes with a single signal using ampliconsto detect the presence of an analyte of interest in a sample. A foodsample 7000 is analyzed to determine the presence or absence ofpathogenic E. Coli. The food sample 7000 is processed (e.g. enriched,cultured, nucleic acid, purification, isolation, extraction, or othersimilar steps) to extract, isolate or otherwise make available thenucleic acids present in the food sample. The nucleic acid sequencespresent in the processed sample 7001 can be amplified, such as but notlimited to by PCR, to amplify the specific pathogenic E. Coli sequences.Examples of these sequences are described herein. No specific primer setneed be used as those can be modified based upon the target sequence tobe amplified. As described herein, the primers can be labeled, therebycreating labeled amplicons (analytes with heterologous interactionunits). The first analyte 7020 and the second analyte 7040 will begenerated if the target sequences are present in the food sample and theprocessed sample. The analytes are shown with heterologous interactionunits (7021, 7022, 7041, and 7042). The analytes can be mixed with abridge unit 7030. The mixture will form a bridged complex 7100. Theanalytes can then be detected by contacting the bridged complex with asolid support 7010 comprising a capture reagent 7015 and a signaldetection unit 7060 comprising a capture reagent 7050. As discussedherein, the a signal detection unit 7060 comprising a capture reagent7050 can be absorbed onto a membrane and allowed to interact with thebridged complex. The solid support 7010 comprising a capture reagent7015 can be a test membrane with an antibody. These elements can beincorporated into a device as described herein. Although FIG. 7 showsthe steps being performed separately they can also be performed indifferent order and some steps may be combined. For example, the step ofmixing the analytes with the bridge unit can also be combined withcontacting the analytes with the signal detection unit comprising acapture reagent. The detection step of adding to the complex to thesolid support could then be done subsequently. In some embodiments, theanalytes, bridge unit, signal interaction unit comprising a capturereagent, and the solid support comprising a capture reagent can be mixedtogether simultaneously or nearly simultaneously and then the signaldetection unit can be detected. The signal detection unit will only bedetected or detected above background levels (i.e. above a negativecontrol) when the plurality of the analytes are present in the samplebeing tested. That is, in FIG. 7, the complex 7200 will only be formedif both analytes, and thus both target sequences are present in the foodsample 7000, are present. The complex 7200 will not be formed if one ofthe analytes is missing. The workflow shown in FIG. 7 can also include awashing step to wash away any unbound material or components that do notform a complex 7200. Washing steps may also be incorporated into anymethod described herein.

In some embodiments of the methods described herein, the method ofdetecting a plurality of analytes with a single signal comprisesamplifying a plurality of target nucleic acid sequences present in asample. The target sequences can be the analytes or the amplifiedproducts can be the analytes. The detection of the amplified sequences(e.g., PCR products) indicates the presence of the template sequences inthe original sample.

In some embodiments, methods of concurrently detecting a plurality ofanalytes with a single signal comprise a) contacting a device fordetecting a plurality of analytes with a single signal with one or moresamples comprising a plurality of analytes; and detecting the presenceor absence of the signal detection unit which indicates the presence orabsence of the first analyte of interest and second analyte of interestconcurrently. The device can be any device used to detect the presenceor absence of analyte including, but not limited to the devicesdescribed herein. In some embodiments, the device comprises: a housingcomprising: an inlet opening in fluid contact with a conjugate pad; aforce member; a slidable locking member contacting the force member; anattachment member contacting the force member; a sliding buttoncontacting the attachment member; and a detection membrane systemcomprising the conjugate pad, a test membrane, and an absorbent member,at least a portion of the conjugate pad, test membrane, and absorbentmember are substantially parallel to each other, the force membercontacts the detection membrane system and is capable of applyingpressure substantially perpendicular to the detection membrane system,the sliding button moves the slidable locking member, the conjugate padcomprises a signal detection unit comprising a third capture reagent;the test membrane comprises a first capture reagent affixed to the testmembrane.

In some embodiments, the one or more samples comprises a first analyteof interest, a second analyte of interest, and a bridge unit comprisinga second capture reagent, wherein the first analyte of interestcomprises a first interaction unit that binds to the first capturereagent and a second interaction unit that binds to the bridge unit, andthe second analyte of interest comprises a first interaction unit thatbinds the bridge unit and a second interaction unit. In someembodiments, the signal detection unit comprises the third capturereagent that binds to the second analyte, to the second analyte's firstinteraction unit or second interaction unit, to a component of the firstand second analyte complex, or to a component of the bridge unit thatthat is only present when the complex contains the first and secondanalytes.

In some embodiments, the detecting comprises moving the conjugate padafter a portion of the one or more samples has contacted and flowedthrough the conjugate pad, thereby exposing at least a portion of thetest membrane for detection of the signal detection unit to indicate thepresence or absence of the plurality of analytes with a single signal.In some embodiments, the conjugate pad is moved by moving the slidablelocking member. In some embodiments, the one or more samples arecontacted with the conjugate pad prior to compressing the detectionmembrane system. The method can be performed with multiple samples todetect the plurality of analytes. For example, if a plurality ofamplification reactions are performed to produce a plurality ofamplicons (analytes) each of the plurality of amplifications reactionsis considered a separate sample. To detect the plurality of analyteswith a single signal the samples have to be mixed. The plurality ofsamples can be mixed prior to contacting the device or be contacted withthe device (solid support) sequentially, or simultaneously.

In some embodiments, the first and second analyte are amplicons. In someembodiments, the first and second analytes are PCR reaction products. Insome embodiments, the first analyte's first interaction unit is adigoxigenin label. In some embodiments, the first analyte's secondinteraction unit is a rhodamine label. In some embodiments, the secondanalyte's first interaction unit is a rhodamine label. In someembodiments, the second analyte's second interaction unit is afluorescein label. In some embodiments, the third capture reagent bindsto the second analyte's second interaction unit. In some embodiments,the third capture reagent is a biotinylated capture reagent. In someembodiments, the signal interaction unit is coated with streptavidin. Insome embodiments, the signal interaction unit is streptavidin coatedcolloidal gold. In some embodiments, the first and second analytes arenucleic acid amplification products, wherein: the first analytecomprises a digoxigenin label and a rhodamine label; the second analytecomprises a rhodamine label and a fluorescein label; the first capturereagent is an anti-digoxigenin label antibody; the second capturereagent is an anti-rhodamine label antibody; the third capture reagentis a biotinylated anti-fluorescein label antibody; and the signalinteraction unit is streptavidin coated colloidal gold.

As used herein and throughout, the terms “attached” or “attachment” caninclude both direct attachment or indirect attachment. Two componentsthat are directly attached to one another are also in physical contactwith each other. Two components that are indirectly attached to oneanother are attached through an intermediate component. For example,Component A can be indirectly attached to Component B if Component A isdirectly attached to Component C and Component C is directly attached toComponent B. Therefore, in such an example, Component A would be said tobe indirectly attached to Component B.

The term “capture reagent” means a reagent capable of binding a targetmolecule or analyte to be detected in a sample. Examples of capturereagents include, but are not limited to, antibodies or antigen bindingfragments thereof, an oligonucleotide, and a peptoid. Other examples ofcapture reagents include, but are not limited to, small molecules orproteins, such as biotin, avidin, streptavidin, hapten, digoxigenin,BRDU, single and double strand nucleic acid binding proteins or otherintercalating agents, and the like, or molecules that recognize andcapture the same. These are non-limiting examples of capture reagents.Other types of capture reagents can also be used.

As discussed herein, a capture reagent can also refer to, for example,antibodies. Intact antibodies, also known as immunoglobulins, aretypically tetrameric glycosylated proteins composed of two light (L)chains of approximately 25 kDa each, and two heavy (H) chains ofapproximately 50 kDa each. Two types of light chain, termed lambda andkappa, exist in antibodies. Depending on the amino acid sequence of theconstant domain of heavy chains, immunoglobulins are assigned to fivemajor classes: A, D, E, G, and M, and several of these may be furtherdivided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1,and IgA2. Each light chain is composed of an N-terminal variable (V)domain (VL) and a constant (C) domain (CL). Each heavy chain is composedof an N-terminal V domain (VH), three or four C domains (CHs), and ahinge region. The CH domain most proximal to VH is designated CH1. TheVH and VL domains consist of four regions of relatively conservedsequences named framework regions (FR1, FR2, FR3, and FR4), which form ascaffold for three regions of hypervariable sequences (complementaritydetermining regions, CDRs). The CDRs contain most of the residuesresponsible for specific interactions of the antibody or antigen bindingprotein with the antigen. CDRs are referred to as CDR1, CDR2, and CDR3.Accordingly, CDR constituents on the heavy chain are referred to as H1,H2, and H3, while CDR constituents on the light chain are referred to asL1, L2, and L3. CDR3 is the greatest source of molecular diversitywithin the antibody or antigen binding protein-binding site. H3, forexample, can be as short as two amino acid residues or greater than 26amino acids. The subunit structures and three-dimensional configurationsof different classes of immunoglobulins are well known in the art. For areview of the antibody structure, see Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Eds. Harlow et al., 1988. One of skill inthe art will recognize that each subunit structure, e.g., a CH, VH, CL,VL, CDR, and/or FR structure, comprises active fragments. For example,active fragments may consist of the portion of the VH, VL, or CDRsubunit that binds the antigen, i.e., the antigen-binding fragment, orthe portion of the CH subunit that binds to and/or activates an Fcreceptor and/or complement.

Non-limiting examples of binding fragments encompassed within the term“antigen-specific antibody” used herein include: (i) an Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii)an F(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) an Fd fragmentconsisting of the VH and CH1 domains; (iv) an Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAbfragment, which consists of a VH domain; and (vi) an isolated CDR.Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they may be recombinantly joined by asynthetic linker, creating a single protein chain in which the VL and VHdomains pair to form monovalent molecules (known as single chain Fv(scFv)). The most commonly used linker is a 15-residue (Gly₄Ser)₃peptide, but other linkers are also known in the art. Single chainantibodies are also intended to be encompassed within the terms“antibody or antigen binding protein,” or “antigen-binding fragment” ofan antibody. The antibody can also be a polyclonal antibody, monoclonalantibody, chimeric antibody, antigen-binding fragment, Fc fragment,single chain antibodies, or any derivatives thereof. The capture reagentor antibody can also be a VHH region, a bi-specific antibody, a peptidefragment comprising an antigen binding site, or a compound that binds toan antigen of interest. The antigen of interest can be an amplicon orother type of analyte.

These antibodies can be purchased or obtained using conventionaltechniques known to those skilled in the art, and the fragments arescreened for utility in the same manner as intact antibodies. Antibodydiversity is created by multiple germline genes encoding variabledomains and a variety of somatic events. The somatic events includerecombination of variable gene segments with diversity (D) and joining(J) gene segments to make a complete VH domain, and the recombination ofvariable and joining gene segments to make a complete VL domain. Therecombination process itself is imprecise, resulting in the loss oraddition of amino acids at the V(D)J junctions. These mechanisms ofdiversity occur in the developing B cell prior to antigen exposure.After antigenic stimulation, the expressed antibody genes in B cellsundergo somatic mutation. Based on the estimated number of germline genesegments, the random recombination of these segments, and random VH-VLpairing, up to 1.6×10⁷ different antibodies may be produced (FundamentalImmunology, 3rd ed. (1993), ed. Paul, Raven Press, New York, N.Y.). Whenother processes that contribute to antibody diversity (such as somaticmutation) are taken into account, it is thought that upwards of 1×10¹⁰different antibodies may be generated (Immunoglobulin Genes, 2nd ed.(1995), eds. Jonio et al., Academic Press, San Diego, Calif.). Becauseof the many processes involved in generating antibody diversity, it isunlikely that independently derived monoclonal antibodies with the sameantigen specificity will have identical amino acid sequences.

Antibody or antigen binding protein molecules capable of specificallyinteracting with the antigens, epitopes, or other molecules describedherein may be produced by methods well known to those skilled in theart. For example, monoclonal antibodies can be produced by generation ofhybridomas in accordance with known methods. Hybridomas formed in thismanner can then be screened using standard methods, such asenzyme-linked immunosorbent assay (ELISA) and Biacore analysis, toidentify one or more hybridomas that produce an antibody thatspecifically interacts with a molecule or compound of interest.

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal antibody to a polypeptide of the present invention may beidentified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) with apolypeptide of the present invention to thereby isolate immunoglobulinlibrary members that bind to the polypeptide. Techniques andcommercially available kits for generating and screening phage displaylibraries are well known to those skilled in the art. Additionally,examples of methods and reagents particularly amenable for use ingenerating and screening antibody or antigen binding protein displaylibraries can be found in the literature.

The term “capture reagent” also includes chimeric antibodies, such ashumanized antibodies, as well as fully humanized antibodies. In someembodiments the capture reagent is a Goat anti-E. coli 0157:H7 antibodyCat #: 70-XG13 (Fitzgerald Industries); E. coli 0157:H7 mono Cat #:10-E13A(Fitzgerald Industries); E. coli 0157:H7 Cat #:10C-CR1295M3(Fitzgerald Industries); E. coli 0157:H7 mono Cat #:10-E12A(Fitzgerald Industries); or Goat anti-mouse IgG Cat #: AB SE-020(DCN). The capture reagent can also be, for example, protein A, proteinG, and the like. The capture reagent can also be an antibody that bindsor specifically binds to a fluorescent label (e.g. fluorescein orrhodamine), a hapten, digoxigenin and the like. A capture reagent, sucha streptavidin can be conjugated with colloidal gold. Thestreptavidin-gold complex can then be used, for example, to bind to abiotinylated product, such as a biotinylated antibody. A non-limitingexample can be seen in FIG. 3. The labels shown in FIG. 3 are forillustrative purposes only and other permutations can be used.

The capture reagent can also include an anti-antibody, i.e. an antibodythat recognizes another antibody but is not specific to an analyte, suchas, but not limited to, anti-IgG, anti-IgM, or anti-IgE antibody.

As used herein, the term “concurrently” refers to the detection ofmultiple analytes simultaneously or nearly simultaneously. As usedherein, “A method of concurrently detecting a plurality of analytes witha single signal,” or variations thereof, refers to a method that uses asingle assay (e.g. single well, single dot, single location on an array)or a single use of a device to detect the plurality of analytes with asingle signal. If different devices, wells, or arrays are used to detectthe plurality of analytes with the same signal this is not a method ofconcurrently detecting a plurality of analytes with a single signal. Fora method to be a method of concurrently detecting a plurality ofanalytes with a single signal the method must generate only a singlesignal (examples of signals are described herein) in a single location(well, dot, line on a membrane or other type of solid support, and thelike), that informs the user that the plurality of analytes are presentin the sample. For example, the same signal being used in differentwells to indicate whether a single analyte is present in that well (orspots on an array) and then analyzing the multiple wells (or spots) todetermine if the plurality of analytes are present is not a method ofconcurrently detecting a plurality of analytes with a single signal.

As used herein, the term “single signal” means detection of a signalbased upon a single moiety or method. For example, if the single signalis the color red, then the plurality of analytes indicated by only uponthe presence of the color red. That is, the color red, in thisnon-limiting example, indicates that the plurality analytes are presentin the sample. In contrast, if one analyte is indicated by the color redand a second analyte is indicated by the color yellow, the use of twocolors (i.e., signals) is not the detection of a plurality of analyteswith a single signal. The signal is not limited to colorimetricdetection. Examples are provided herein of signals that can be used.

The term “detecting” or “detection” is used in the broadest sense toinclude qualitative and/or quantitative measurements of a targetanalyte.

As used herein, the term “interaction unit” means a part of the analyteor a heterologous tag or label that is attached to the analyte that thatis recognized or bound by another molecule (e.g. the capture reagent,the bridge unit, or the signal detection unit). The interaction unit canbe part of the analyte itself or can be a heterologous tag or label. Theinteraction unit can also be an antibody or other type of capturereagent that recognizes the analyte. In some embodiments, the analytecan comprise more 1, 2, 3, 4, or 5, interaction units. In someembodiments, the interaction unit is a capture reagent that binds toanother interaction unit present on the analyte itself or a heterologoustag that is part of the analyte. For example, if the analyte is apeptide or part of a protein, a part of the protein or peptide itselfcan be the interaction unit recognized by the capture reagent, bridgeunit, or the signal detection unit. In some embodiments, the peptide canalso be covalently attached to a heterologous tag or label and theheterologous tag or label or the complex of the peptide with theheterologous tag or label is considered the interaction unit. Thus, insome embodiments, an analyte comprises a first interaction unit and/or asecond interaction unit. In some embodiments, the interaction unit(s)can be intrinsic to the analyte itself or the interaction unit(s) couldbe added through some other method, such as cross-linking, covalentattachment through a chemical reaction, non-covalent interactions (e.g.antibody-antigen, hybridization between a part of the analyte andanother molecule, and the like). Where the interaction unit is formed bythe hybridization of two molecules (e.g. two nucleotide sequences), suchthat the part of the hybridization product that is recognized by anothermolecule would be considered the interaction unit. The interaction unitscan also be added to the analyte through an amplification reaction. Thiscan be produced through the use of primers that contain the interactionunits. Interaction units can also have detectable signals, but it is notthese signals that are detected.

As used herein, the term “heterologous” in reference to the interactionunit means a group, molecule or moiety that is not native to theanalyte. For example, an amplification product can comprise just nucleicacid molecules or nucleotide bases. The amplification product, however,can be conjugated to or attached to a heterologous tag, such as, but notlimited to, hapten, biotin, digoxigenin, a fluorescent molecule (e.g.fluorescein or rhodamine) and the like. Examples of heterologousinteraction units include, but are not limited to, hapten, biotin,nucleic acid molecules, peptide fragments (e.g. His-tags, GST-tags, andthe like), enzymes, streptavidin, avidin, fluorescent molecules, and thelike. This list is non-limiting and any interaction unit can be used.Analytes can be labeled with molecules such as digoxigenin, rhodamine,fluorescein, DNP, BRDU, and then be detected by capture reagents thatare specific for a given molecule.

According to some embodiments, methods of detecting a plurality ofanalytes is provided. The presently described methods can be used todetect multiple analytes. An unexpected and surprising result is thatmultiple analytes can be detected using a single signal. This has theunexpected result that the presence, or absence, of multiple analytescan be detected with only the detection of one signal. This is incontrast to the detection of the presence of multiple analytes usingdistinct signals in the same reaction to detect the presence of multipleanalytes in a sample or requiring the performing of separate reactionsand methods to detect multiple analytes. That is, the embodimentsdescribed herein provide, in part, methods of detecting multipleanalytes concurrently with a single signal, such that the detection of asingle signal indicates the presence of the multiple analytes in asample or that the absence of the single signal indicates the absence ofthe multiple analytes in the sample. The present embodiments providesmethods of detecting at least 2, 3, 4, or 5 analytes concurrently with asingle signal. In some embodiments, the method can be used to detect 2,3, 4, or 5 different analytes concurrently with a single signal.Although many examples are provided for detecting 2 analytes, themethods can be adapted and modified based upon the present disclosurefor the detection of 3, 4, or 5 analytes.

As used herein, the term “different analytes” means the analytes are notthe same. The different analytes, however, can be referred to with thesame name, but be from different organisms or from different strains ofthe same organism. For example different organisms contain genes andproteins that have the same function and, therefore, have been given thesame name. But the genes or proteins are from different sources and thusare considered different analytes. They may or may not have differentsequences. Different analytes can also means analytes from differentorganisms. For example, there are any many strains of E. coli. Not allstrains of E. coli cause a food-borne illness. The present methods canbe used, for example, to detect a plurality of analytes from apathogenic E. coli strain as opposed to detecting an analyte from anon-pathogenic E. coli strain. Although reference made be madethroughout the present disclosure to specific types of analytes, theanalytes can be any type of analyte, such as but not limited, to theclasses of analytes described herein.

For example, in some embodiments, methods of concurrently detecting afirst analyte and a second analyte are provided. In some embodiments,the method comprises contacting a solid support, which comprises a firstcapture reagent with a first analyte, a second analyte, a bridge unit,which comprises a second capture reagent, and a signal detection unitcomprising a third capture reagent; and detecting the presence of thesignal detection unit which indicates the presence of the first analyteand second analyte. In some embodiments, the first capture reagent isaffixed to the solid support. In some embodiments, the first analytecomprises a first interaction unit that binds to the first capturereagent and a second interaction unit that binds to the bridge unit; thesecond analyte comprises a first interaction unit that binds the bridgeunit and a second interaction unit that binds to the signal detectionunit. The signal detection unit can then be detected. If the signaldetection unit is detected, it indicates that the multiple analytes arepresent.

Without desiring to be bound by any theory, the multiple analytes can bedetected concurrently by forming a complex. In some embodiments, thecomplex comprises the solid support, the first analyte, the secondanalyte, the bridge unit, and the signal detection unit wherein eachmember of the complex binds to each other directly or indirectly. Thesample can be washed while retaining the solid support and the complexwill only be detected if the complex is formed. Examples of thesecomplexes can be seen in FIGS. 1-3, which are further described herein.

In some embodiments, methods of concurrently detecting a first analyteand a second analyte are provided, the method comprising: contacting asolid support with a first analyte of interest, a second analyte ofinterest, a bridge unit comprising a second capture reagent, and asignal detection unit comprising a third capture reagent; and detectingthe presence or absence of the signal detection unit which indicates thepresence or absence of the first analyte of interest and second analyteof interest concurrently, wherein: a first capture reagent is affixed tothe solid support; the first analyte of interest comprises a firstinteraction unit that binds to the first capture reagent and a secondinteraction unit that binds to the bridge unit; and the second analyteof interest comprises a first interaction unit that binds the bridgeunit; a signal detection unit that binds to the second analyte, to thesecond analyte's first interaction unit or a second interaction unit, toa component of the first and second analyte complex or bridge unit thatthat is only present when the complex contains the first and secondanalyte.

FIG. 1 illustrates a complex that could be formed to detect two analytesconcurrently with a single signal. FIG. 1 illustrates a capture reagent15 affixed to a solid support 10. The capture reagent 15 binds to thefirst analyte 20. The bridge unit 30 binds to the first analyte. Thebridge unit also binds to the second analyte 40. FIG. 1 also illustratesa signal detection unit 60 comprising a capture reagent 50 that binds tothe second analyte 40. FIG. 1 illustrates that this complex only formswhen all of the members are present and can bind to one another. Thesignal detection unit can then be detected. FIG. 3 also shows anembodiment of detecting two analytes with a single signal. FIG. 3 showsspecific labels (e.g. FITC, TAMRA, DIG, biotin, streptavidin, etc. . . .), but these labels can be modified according to the present disclosure.

In some embodiments, the method comprises one or more washing steps. Thewashing step can be used to remove unbound materials. For example, ifthe solid support is contacted with a first sample, the solid supportcan be washed to remove any unbound material. In some embodiments wherethe solid support is a bead, the beads can be contacted with the sampleand then the beads can be washed. Washing beads is routine and wellknown to one of skill in the art. The method of washing beads or othertypes of solid supports can be altered or chosen based upon the specificsolid support that is used and is often not a critical feature.

In some embodiments, the sample with the first analyte is contacted withthe solid support. In some embodiments, the mixture is washed such thatany materials not bound to the solid support are no longer present. Insome embodiments, the solid support is also contacted with the samesample or a different sample comprising the second analyte and/or thebridge unit. The mixture can then be washed again to remove any unboundmaterial. In some embodiments, a signal detection unit comprising acapture reagent is added. A washing step can also be included to removeany unbound signal detection units. The signal detection unit can thenbe detected or another reagent can be added that detects the presence ofthe signal detection unit. In some embodiments, all of the steps areperformed simultaneously or nearly simultaneously. During theperformance of the method, a washing step may be inserted whereappropriate.

In some embodiments, the different analytes or samples can be mixedtogether before or simultaneously applied to the solid support. Thesamples can be, for example, amplification reaction mixtures that wereused to produce, or attempted to produce, the analytes. In someembodiments, different amplification reactions will be performed toamplify the plurality of analytes. Therefore, prior to the samples oranalytes being applied to the solid support the samples or analytes canbe mixed together. The samples or analytes can also be mixed with thecapture reagents and/or bridging units prior to being contacted with thesolid surface.

In some embodiments, the first and second interaction unit of the firstanalyte and the first and second interaction unit of the second analyteare each independently a heterologous interaction unit. In someembodiments, an interaction unit of the first analyte and an interactionunit of the second analyte is a hapten. In some embodiments, theinteraction unit of the first analyte and the second analyte isfluorescein or rhodamine molecule. Accordingly, in some embodiments, thefirst analyte and second analyte have at least one interaction unit incommon. The commonality of the interaction unit will enable the bridgingunit to bring the two analytes into a detectable complex. In someembodiments, the first and second analytes do not have the sameinteraction unit. In such a case for some embodiments, the bridgingentity would be a bivalent capture reagent (e.g. bivalent antibody) thatcan link the two analytes to one another. The bivalent capture reagentwould be able to bind to both the first and second analytessimultaneously. In some embodiments, each of the interaction unitspresent on the plurality of analytes are different. In some embodiments,some of the interactions units are different, but some of theinteraction units are the same. In some embodiments, the analytecomprises a hapten interaction unit and a biotin interaction unit. Insome embodiments, the first analyte comprises a digoxigenin interactionunit and a rhodamine interaction unit; the second analyte comprises arhodamine interaction unit and a FITC interaction unit, and the bridgingunit binds to the rhodamine interaction unit. The bridging unit can thenform the complex that contains both the first and second analyte. Thiscan be seen, for example, in FIG. 3.

In some embodiments, the plurality of the analytes are the same type ofanalyte. For example, each of the analytes being detected can be apeptide. In some embodiments, each of the analytes is a nucleic acidmolecule, such as an amplification product (e.g. amplicon). The analytescan also be any type, including, but not limited to, the analytesdescribed herein. In some embodiments, the analytes are different. Insome embodiments, a first analyte is an amplification product and asecond analyte is a protein or peptide. Any combination of analytes canbe used.

As used herein, the term “bridge unit” or “bridge” means a molecule(s)that can link two or more analytes in a complex. That is, for example,the bridge unit can bind to an interaction unit on a first analyte andan interaction unit on a second analyte. If only detecting two analytes,one bridge unit may be used. If detecting three analytes, two bridgeunits can be used. In some embodiments, the methods use “n−1” bridgeunits, where “n” is the number of analytes being detected. In someembodiments, a single bridge unit is used to detect more than 2analytes. Examples of bridge units include, but are not limited to,immunoglobulin molecules (e.g. IgM, IgE, IgG, IgA, and the like),streptavidin, and a molecule that comprises a plurality of capturereagents such that the bridge unit can bind to more than one interactionunit. In some embodiments, the bridge unit is a multivalent capturereagent.

In some embodiments, the bridge unit is a complex of compounds,substances, or macromolecules. For example, a bridge unit could comprisea nanoparticle coated with antibodies and a separate antibody. In thisnon-limiting example, the nanoparticle coated with antibodies cancontain antibodies that bind to an analyte or interaction unit on theanalyte and also contain antibodies that bind to the separate antibody.The separate antibody can bind to a different analyte. The interactionof the nanoparticle coated with antibodies and the separate antibodywould then be able to bridge together the different analytes. Anon-limiting illustration of this bridge complex can be seen in FIGS. 4and 5, which is also described below. Other variations of the bridgebeing a complex could also be made. The exact structure and form of thebridge unit is not essential so long as it can “bridge” a plurality ofanalytes in a complex. Thus, the bridge could be made up of multiplesubunits or components to bridge the analytes together. Although thebridge unit can be illustrated and discussed bridging two analytes, thebridge unit can be designed to bridge more than 2 analytes, such as 3,4, 5, or more. Therefore, in some embodiments, the bridge unit bridges2, 3, 4, 5, or more analytes. In some embodiments, the bridge unitbridges at least 2, 3, 4, or 5 analytes. The non-limiting example ofbridging 2 analytes is for illustrative purposes only and theembodiments disclosed herein are not limited to bridging only 2analytes.

As discussed herein, the present methods can be applied to detectingmore than 2 analytes. For example, a method of detecting a firstanalyte, a second analyte, and a third analyte concurrently with asingle signal is provided. For the detection of additional analytes, themethods can be adapted in a similar manner. In some embodiments, themethod comprises contacting the first, second, and third analytes with asolid support, a first bridge unit, a second bridge unit, and a signaldetection unit; and detecting the presence of the signal detection unitwhich indicates the presence of the first, second, and third analytesconcurrently with a single signal, wherein the first analyte comprises afirst interaction unit and a second interaction unit; the second analytecomprises a first interaction unit and a second interaction unit; thethird analyte comprises a first interaction unit and a secondinteraction unit; the solid support comprises a first capture reagentthat binds to the first interaction unit of the first analyte; the firstbridge unit binds to the second interaction unit of the first analyteand the first interaction unit of the second analyte; the second bridgeunit binds to the second interaction unit of the second analyte labeland the third interaction unit of the third analyte; and the signaldetection unit binds to the second interaction unit of the thirdanalyte. Without being bound to any theory, it is expected that theanalytes can be concurrently detected because the first, second, andthird analytes form a complex, wherein the complex comprises the solidsupport, the first analyte, the second analyte, the third analyte, thefirst bridge unit, the second bridge unit, and the signal detection unitwherein each member of the complex binds to each other directly orindirectly.

FIG. 2 illustrates a complex that can be formed to detect threeanalytes, which is analogous to the example illustrated in FIG. 1. FIG.2 illustrates a solid support 10 with a capture reagent 15 bound to afirst analyte 70. The first analyte is bound to a first bridge unit 80.The first bridge unit is bound to a second analyte 20, which is alsobound to a second bridge unit 30. The second bridge unit is also boundto a third analyte 40, which is bound to a capture reagent 50. Thecapture reagent is also attached to a signal detection unit 60. Thus,FIG. 2 illustrates a non-limiting example of how three analytes can bedetected with a single signal.

FIG. 4 illustrates a non-limiting bridge complex made up of more thanone molecule, macromolecule, or substance. This can be referred to as amulti-component bridge complex. FIG. 4 illustrates a bridge unit 30 thatcomprises a particle 34, a first capture reagent 31, a second capturereagent 32, and a third capture reagent 33. The bridge unit 30 is ableto bring together the first analyte 20 and the second analyte 40 andfrom a complex linking the first analyte 20 and the second analyte 40.FIG. 4 illustrates a particle 34 (e.g. nanoparticle, polystyrene,agarose, and the like) coated with a first capture reagent 31 that bindsto the first analyte 20, either directly or indirectly through aninteraction unit, a third capture reagent 33, which is also present onthe particle 34, that binds to a second capture reagent 32 that is boundto the second analyte 40. This complex can then be detected according tothe methods and compositions described herein, which is illustrated inFIG. 5. FIG. 5 shows the bridges complex of FIG. 4 interacting with asolid support 10 with a capture reagent 15 bound to a first analyte 20and a signal detection unit 60 comprising a capture reagent 50 thatbinds to the second analyte 40. As discussed herein, the illustration ofthe signal detection unit binding to the second analyte is forillustrative purposes only. The signal detection unit can also bindother parts of the complex so long as the signal detection unit is notbinding to the analyte that is interacting with the solid support or thesolid support itself. The solid support 40 can be, for example, a testmembrane, such as the test membrane that is shown in FIG. 3. Otherexamples of solid supports are provided herein.

The present invention provides complexes comprising a solid support, afirst analyte, a second analyte, a bridge unit, and a signal detectionunit wherein each member of the complex binds to each other directly orindirectly. In some embodiments, the solid support is bound to the firstanalyte, the bridge unit is bound to the first analyte and the secondanalyte, and the signal detection unit is bound to the second analyte.In some embodiments, the solid support comprises a first capturereagent, the first analyte comprises a first interaction unit and asecond interaction unit, the second analyte comprises a firstinteraction unit and a second interaction unit, the bridge unitcomprises one or more capture reagents that independently bind to thesecond interaction unit of the first analyte and the first interactionunit of the second analyte, and the signal detection unit comprises acapture reagent that binds to the second interaction unit of the secondanalyte.

In some embodiments, the complex comprises a solid support, a firstanalyte, a second analyte, a third analyte, a first bridge unit, asecond bridge unit, and a signal detection unit, wherein the solidsupport, the first analyte, second analyte, third analyte, first bridgeunit, second bridge unit, and signal detection unit are bound to eachother directly or indirectly. In some embodiments, the solid supportbinds to the first analyte, the first bridge unit binds to the firstanalyte and the second analyte, the second bridge unit binds to thesecond analyte and the third analyte, and the signal detection unitbinds to the third analyte. In some embodiments, the solid supportcomprises a first capture reagent, the first analyte comprises a firstinteraction unit and a second interaction unit, the second analytecomprises a first interaction unit and a second interaction unit, thethird analyte comprises a first interaction unit and a secondinteraction unit, the first bridge unit comprises one or more capturereagents that independently bind to the second interaction unit of thefirst analyte and the first interaction unit of the second analyte, thesecond bridge unit comprises one or more capture reagents thatindependently bind to the second interaction unit of the second analyteand the first interaction unit of the third analyte, and the signaldetection unit comprises a capture reagent that binds to the secondinteraction unit of the third analyte.

In some embodiments, the presently described methods can be used todetect a food borne pathogen by the detection of a plurality offood-borne pathogen analytes with a single signal. For example, a samplemay be processed to isolate an analyte (e.g. an antigen or a toxin, or afood borne pathogen nucleic acid may be isolated or amplified). Theplurality of analytes (e.g. food borne pathogen protein and/or anamplicon) can be detected concurrently with the methods describedherein. The methods can then provide greater confidence in thespecificity of the test and avoid false negatives. In some embodiments,a positive result that indicates the presence of a food borne pathogenrequires the detection of 2, 3, or 4 analytes. The present methods canbe used to detect the analytes concurrently with a single signal. Thesingle signal provides an easier result to interpret since the signalwill only be detectable if all of the plurality of analytes beingdetected are present in the sample. Thus, if 2 analytes are beingdetected then the signal will only be detectable if both analytes arepresent. In some embodiments, the signal is only detectable when 3analytes are present. This type of methods can be applied to othermethods of detection.

In some embodiments, the method can be used to detect 3 classes ofanalytes to provide a positive test for food contamination. In someembodiments, one of the analytes is a toxin (e.g. Shiga toxin 1 and/or2). The toxin can be detected itself or the nucleotide sequence thatencodes or controls the production of the toxin can be detected. In someembodiments, one of the analytes is eae gene, which can also be referredto as a virulence factor. The eae gene can be found, or expressed in,for example, enterohemorrhagic Escherichia coli.

In some embodiments one of the analytes is a serotype analyte, which canbe an antigen that is specifically produced by a strain of a food bornepathogen. In some embodiments, the serotype analyte is an E. coliserotype. In some embodiments, the E. coli serotype is O26, O45, O103,O111, O121, and O145. Therefore, in some embodiments, a positive testfor food borne contamination requires the detection of 3 analytes with asingle signal, wherein the 3 analytes are the Shiga toxin (e.g. Shigatoxin 1 and/or 2), the eae gene, and a serotype analyte chosen from E.coli serotype is O26, O45, O103, O111, O121, and O145. In someembodiments, the serotype analyte is a protein specifically expressed bya pathogenic strain. In some embodiments, the analyte is a nucleic acidsequence that encodes the antigen. In some embodiments, the nucleic acidsequence is a fragment of the coding sequence of the antigen. Thespecific fragment is not critical and one of skill in the art candetermine the sequences or fragments thereof to amplify using routinemethods. As discussed herein, the target sequence can be amplified andoptionally labeled with a heterologous interaction unit. The analytescan then be detected according to the methods provided herein.

For example, if a positive test for a virus requires the presence of twodistinct nucleic acid sequences, the two nucleic acid sequences can bedetected concurrently with a single signal using the methods describedherein as opposed to detecting the two nucleic acid sequences separatelywith more than one signal. Additionally, if the presence of cancerrequires the detection of a plurality of genes being expressed insample, the genes can be detected concurrently with a single signal byusing analytes that correlate with their expression (e.g. by usingRT-PCR to amplify the gene product) according to a method describedherein. Therefore, the presently described methods have wideapplicability and can be used with any plurality of analytes (targetmolecules) and even with analytes that are not the same.

In some embodiments, methods are provided for detecting two or moreanalytes comprising detecting the multiple analytes using a flow(vertical or lateral) device. Examples of vertical flow devices andmethods of using them are provided in U.S. Pat. Nos. 8,012,770,8,183,059 and U.S. patent application Ser. Nos. 13/500,997, 13/360,528,13/445,233, each of which is hereby incorporated by reference in itsentirety. The devices can be adapted for the detection of multipleanalytes using a single signal.

Accordingly, embodiments provided herein provide methods of detectingmultiple analytes with a single signal by using vertical flow anddevices employing vertical flow. Vertical flow allows the analyte and/orthe sample to flow through the layers/membranes of the analyte detectionmembrane system. By “through layers” or “through membranes” is meant torefer to the sample flowing through the layers and vertically across thelayers. In some embodiments, the sample does not flow horizontally orlaterally across the different layers/membranes.

The following terms are used in conjunction with the description ofvarious vertical flow devices. Other terms are defined relevant to somevertical flow devices or uses thereof are described throughout as well.

The term “pressure actuator” and “force actuator” can be usedinterchangeably and refer to a component that can exert, for example,pressure through the application of force. A force actuator can also bereferred to as a force member. Examples of include, but are not limitedto, various force members that are described herein. Other examplesinclude, but are not limited to, pistons or other solid supportstructures. The force actuator's position relative to another componentcan be raised, lowered, or moved laterally. The position of the forceactuator can be controlled manually or through a signal processing unit(e.g. computer). The ability to control the position of the forceactuator can be used to regulate the force (e.g. pressure) being appliedto another component, such as, but not limited to, an analyte detectionmembrane system. By regulating the force applied to the membrane systemthe flow rate of the sample can be regulated. The force can be used tokeep the flow rate of the sample through the membrane system constant orthe flow rate can be variable. The flow rate can also be stopped andallow the sample to dwell on different layers of the membrane system.For example, the sample's flow rate can be zero or near zero when thesample contacts the conjugate pad. After resting on the conjugate padthe flow rate can be increased by modulating the pressure being appliedby the force actuator. The sample can then through the entire membranesystem, or the force being applied can be modulated to allow the sampleto dwell (rest) on another layer of the membrane system. Force can beprecisely regulated, either manually or by using a signal processingunit (e.g. computer) the flow rate can be modified at any point as thesample vertically flows through the membrane system. The flow rate canalso be regulated based upon the absorbency of the membranes in themembrane system and/or the number of the membranes of the system. Basedupon the absorbency the flow rate can be modulated (e.g. increased ordecreased).

The flow rate can be measured in any units including but not limited toμl/min or μl/sec, and the like. The flow rate during a dwell can be, forexample, 0 μl/sec, or less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3,0.2, or 0.1 μl/sec or μl/min. The flow rate can be monitored manually orby a signal processing unit (e.g. computer) and regulated by the same.The flow rate can be regulated and monitored by well-known and routinemethods known to one of skill in the art in addition to those describedherein. In some embodiments, the flow rate is about 0 to 1 ml/min, about0-10 ml/min, about 1-9 ml/min, about 1-8 ml/min, about 1-7 ml/min, about1-6 ml/min, about 1-5 ml/min, about 1-4 ml/min, about 1-3 ml/min, about1-2 ml/min, about 0.5-1.5 ml/min, about 1-1.5 ml/min, or about 0.5-1ml/min. In some embodiments, the flow rate is about 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 ml/min. In some embodiments, the flow rate is at least 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 ml/min. In some embodiments, the flow rate is1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ml/min.

In some embodiments, the devices used to detect multiple analytes with asingle signal comprise a housing comprising a first housing member and asecond housing member. In some embodiments, the first and second housingmembers can be constructed as a single unit. The housing can comprise aninlet opening. The inlet opening allows the introduction of a sampleonto the chromatographic assay. In some embodiments, the first housingmember comprises the inlet opening. The inlet opening can be ofsufficient size to handle an appropriate amount of volume of a solutionthat is added to the device. In some embodiments, the size of theopening is large enough to handle about 0.1 to 3 ml, about 0.1 to 2.5ml, about 0.5 to 2.0 ml, about 0.1 to 1.0 ml, about 0.5 to 1.5 ml, 0.5to 1.0 ml, and 1.0 to 2.0 ml.

In some embodiments, the housing comprises a conjugate pad, a permeablemembrane, a test membrane, and/or an absorbent member. In someembodiments, the housing comprises an analyte detection membrane system.In some embodiments, the analyte detection membrane system comprises aconjugate pad, a permeable membrane, a test membrane, and an absorbentmember. In some embodiments, the analyte detection membrane system isfree of a permeable membrane. In some embodiments, the analyte detectionmembrane system comprises in the following order: a conjugate pad, apermeable membrane, a test membrane, and an absorbent member.

As used herein, the term “conjugate pad” refers to a membrane or othertype of material that can comprise a capture reagent. The conjugate padcan be a cellulose acetate, cellulose nitrate, polyamide, polycarbonate,glass fiber, membrane, polyethersulfone, regenerated cellulose (RC),polytetra-fluorethylene, (PTFE), Polyester (e.g. PolyethyleneTerephthalate), Polycarbonate (e.g., 4, 4-hydroxy-diphenyl-2,2′-propane), Aluminum Oxide, Mixed Cellulose Ester (e.g., mixture ofcellulose acetate and cellulose nitrate), Nylon (e.g., Polyamide,Hexamethylene-diamine, and Nylon 66), Polypropylene, PVDF, High DensityPolyethylene (HDPE)+nucleating agent “aluminum dibenzoate” (DBS) (e.g.80 u 0.024 HDPE DBS (Porex)), and HDPE. Examples of conjugate pads alsoinclude, Cyclopore® (Polyethylene terephthalate), Nucleopore®(Polyethylene terephthalate), Membra-Fil® (Cellulose Acetate andNitrate), Whatman® (Cellulose Acetate and Nitrate), Whatman #12-S(rayon)), Anopore® (Aluminum Oxide), Anodisc® (Aluminum Oxide),Sartorius (cellulose acetate, e.g. 5 μm), and Whatman Standard 17 (boundglass). The conjugate pad can also be made of a material that dissolvesafter coming into contact with a sample or other liquid. The dissolvingof the conjugate pad can be performed so that other layers of thesystems described herein can be revealed or exposed for either visualinspection (e.g. detection of an analyte) or for spectrometer inspection(e.g. detection of an analyte by a spectrometer).

In some embodiments, the conjugate pad or test membrane comprises acapture reagent. In some embodiments, the conjugate pad or test membraneis contacted with the capture reagent and then allowed to dry beforebeing used in the vertical flow device. The conjugate pad or testmembrane can also comprise other compositions to preserve the capturereagent such that it can be stably stored at room temperature or underrefrigeration or freezing temperatures. In some embodiments, theconjugate pad or test membrane is soaked with a buffer prior to thecapture reagent being applied. In some embodiments, the buffer is ablocking buffer that is used to prevent non-specific binding. In someembodiments, the buffer comprises Borate, BSA, PVP40 and/or Tween-100,or any mixture thereof. In some embodiments, the buffer is 10 mM Borate,3% BSA, 1% PVP40, and 0.25% Tween-100. In some embodiments the capturereagent is applied to the pad or membrane in a solution comprisingtrehalose and sucrose. In some embodiments, the capture reagent isapplied to the pad, membrane, or both, in a solution comprisingtrehalose, sucrose and phosphate and/or BSA. In some embodiments, thecapture reagent is applied in a solution that is 5% trehalose, 20%sucrose, 10 mM phosphate, and 1% BSA. In some embodiments, the testmembrane comprises a capture reagent that binds to a labeled amplicon.In some embodiments, the capture reagent is an antibody that recognizesor binds to digoxigenin, fluorescein (e.g. FITC), rhodamine (TAMRA), andthe like.

In some embodiments, the conjugate pad comprises streptavidin. Thestreptavidin can also be further labeled as described herein. In someembodiments, the streptavidin is the capture reagent that binds to abiotinylated antibody that is used to detect multiple analytes with asingle signal.

In some embodiments, the removable member contacts a first surface ofthe conjugate pad and the adhesive member contacts a second surface ofthe conjugate pad.

In some embodiments, the device comprises an adhesive member. Theadhesive member can comprises an adhesive member inlet that allows thesample to flow through the conjugate pad and contact the test membrane.In some embodiments, the adhesive member inlet is the same size or shapeas the removable member inlet. In some embodiments, the adhesive memberinlet is a different size or shape as the removable member inlet. Insome embodiments, the inlets in the adhesive member are the same shapebut have different areas. Inlets with different areas would beconsidered to have different sizes. The adhesive member can be made upof any substance suitable for adhering one member or membrane to anothermember or membrane. In some embodiments, the adhesive member isimpermeable to liquid. In some embodiments, the adhesive member contactsthe removable member.

In some embodiments of the device, the permeable membrane is attached toor adhered to a test membrane. In some embodiments, the permeablemembrane is laminated onto the test membrane. The permeable membrane canbe a membrane of any material that allows a sample, such as a fluidsample, to flow through to the test membrane. Examples of test membraneinclude, but are not limited to, nitrocellulose, cellulose, glass fiber,polyester, polypropylene, nylon, and the like. In some embodiments, thepermeable membrane comprises an opening. The opening can be present toallow visualization or detection of the test membrane. In someembodiments, the opening in the permeable membrane is substantially thesame size as the inlet opening in the housing. Examples of permeablemembranes include, but are not limited to, Protran BA83, Whatman, andthe like.

As discussed herein, one example of a solid support is a test membrane.As used herein and throughout, the “test membrane” refers to a membranewhere detection of a binding partner to a capture reagent occurs. Testmembranes include, but are not limited to a nitrocellulose membrane, anylon membrane, a polyvinylidene fluoride membrane, a polyethersulfonemembrane, and the like. The test membrane can be any material that canbe used by one of skill in the art to detect the presence of a capturereagent's binding partner (e.g. labeled analyte, antigen or epitope).The test membrane can also comprise a capture reagent. In someembodiments, the test membrane is contacted with a capture reagent andthe capture reagent is allowed to dry and adhere to the test membrane.Examples of test membranes include, but are not limited to Protran BA83,Whatman, Opitran BA-SA83, and 0.22 μm white plain (Millipore Product No.SA3J036107). Test membranes may also be comprised of nanoparticlematrices to which capture reagents are bound. Nanocrystals can bearranged into 2D sheets and 3D matrices with materials such as, but notlimited to, carbon based particles, gold or metal alloy particles,co-polymer matrices, as well as monodisperse semiconducting, magnetic,metallic and ferroelectric nanocrystals. The test membrane can comprisea plurality of capture reagents. In some embodiments, the test membranecomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 capture reagents. In someembodiments, the test membrane comprises a plurality of areas each witha different capture reagent. In some embodiments, the plurality of areasdo not completely overlap or coincide with one another.

In some embodiments, the device or housing also comprises an absorbentmember. The absorbent member can also be referred to as a “wick pad” or“wicking pad.” The absorbent member absorbs the fluid that flows throughthe device when the sample is applied to the device and provides for thewicking force that aids in the flow of the sample when it is applied tothe device. “Absorbent member” is meant to refer to a material that hasa capacity to draw (wick) and retain solution away from a surface thatthe material is in contact with. Use of a combination of material ofincreasing or decreasing absorbance can also allow for control of samplemovement.

The absorbent member can be any material that can facilitate the flow ofthe sample through the conjugate pad and to the test membrane. Examplesof absorbent members include, but are not limited to cellulose, superabsorbent polymers, glass fiber pads (e.g. C083 (Millipore)), and thelike. In some embodiments, the housing comprises a plurality (e.g. 2 ormore) of absorbent members. In some embodiments, the housing comprises2, 3, 4, or 5 absorbent members. In some embodiments, the devicecomprises one absorbent member. In some embodiments, the absorbentmember comprises one or more membranes up to 10 individual membranes,and each membrane may be the same material or a different material. Insome embodiments, the device consists of only 1 membrane that is anabsorbent member.

In some embodiments, the device comprises a force member. Examples offorce members are described below and can be seen in the drawings. Theseexamples are non-limiting and other forms of force members can be used.The force member can, in some embodiments, be used to apply pressure orto compress the other components of the analyte detection membranesystem against one another. In some embodiments, the force member can bemade out of any material including, but not limited to plastic orstainless steel. As shown in FIG. 23, clips can act as force members.The stainless steel can be laser cut such that it can act as a clip.Non-limiting examples of these clips can be seen in FIG. 23. The forcemember acts to apply pressure to the membrane system. The force memberis not limited to a clip, but rather can be any shape (see, Figures fornon-limiting examples) that can apply pressure to the membrane system(e.g. nanoparticle matrices) and piston like structures strategicallyplaced within the assembly. In some embodiments, the force member is apiston. The force member can be used to apply pressure or to compressthe other components of the analyte detection membrane system againstone another. In some embodiments, the force member can comprise a shaftand a head. The force member can have a mushroom type shape where thehead is wider than the shaft. In some embodiments, the head is narrowerthan the shaft. The force member comprising a head and a shaft can be asingle unit or can be made up of multiple parts that contact one anotherto form the force member. For example, the head could be one unit thatcan be separated from the shaft. Upon assembly the head and shaft arecontacted with one another to make the force member. In another example,the head and shaft are one cohesive unit and are manufactured togetherand not as separate parts that are later assembled to form the forcemember. The force member allows the device to work with vertical flow asopposed to relying upon lateral flow.

In some embodiments, the force member contacts a surface of theabsorbent member. In some embodiments, the force member contacts asurface of the absorbent member and a surface of the removable layer. Insome embodiments, the force member compresses the membrane detectionsystem from above and below the membrane detection system. For example,in some embodiments, the force member can sandwich all the layers of themembrane detection system. In some embodiments the force member isattached to a support member.

In some embodiments, the device comprises, in the following order, aremovable member, a conjugate pad, and an adhesive member.

The device can also comprise a support member. The support member, insome embodiments, contacts a surface of the absorbent member. Thesupport member can also have a support member inlet. The inlet can bethe same size and/or shape as the inlet in the removable member and/orthe adhesive member. In some embodiments, the support member comprisesan inlet that is a different size and/or shape as the inlet in theremovable member and/or the adhesive member. The support member can bemade from any material including, but not limited to, plastic. In someembodiments, the second housing member serves as the support member.

The devices described herein can be used in assays to detect thepresence of a capture reagent's binding partner. These assays can asshown herein be used to detect multiple analytes for the detection ofsingle signals. For example, an antigen can be detected by an antibodyusing the devices of the present invention. The term “Vertical flow” isused throughout. The term “vertical flow” refers to the direction thatthe sample flows across the different membranes and members present in adevice. Vertical flow refers to a sample flowing through the membrane(e.g. top to bottom) as opposed to lateral flow, which refers to asample flowing across (e.g. side to side) a membrane, pad or absorbentmember. In a lateral flow device the membranes and pads sit horizontallyadjacent to one another substantially on the same plane. In a verticalflow device each membrane or pad is substantially parallel or completelyparallel to each other and occupy substantially different spatial planesin the device. The membranes and pads may occupy similar planes whenthey are compressed or put under pressure. In some embodiments, at leasta portion of each member, membrane, or pad is layered on top of eachother. In some embodiments, at least a portion of each layer of member,membrane, or pad is substantially parallel to each other. In someembodiments, at least a portion of each layer is in a different spatialplane than each other layer.

To allow vertical flow to occur efficiently, in some embodiments andwhen present, the conjugate pad, permeable membrane, test membrane andthe absorbent member are substantially parallel to each other. In someembodiments, the conjugate pad, permeable membrane, test membrane andthe absorbent member are present in different spatial planes. In someembodiments, the housing also comprises a hydrophobic membrane that canslow or stop the vertical flow of the sample. The hydrophobic membranecan be in contact with the test membrane, which would allow the sampleto dwell or rest upon the test membrane. The dwell can allow forincreased sensitivity and detection. The vertical flow is modulated bythe pressure that is applied to the membranes, pads, and/or members. Insome embodiments, the pressure is applied perpendicular to the testmembrane and/or the conjugate pad. In some embodiments, the pressure canbe applied so that the conjugate pad is compressed against the housing.The compression against the housing can be such that the conjugate is indirect contact with the housing, O-ring, or collar, or through anintermediate so that the conjugate pad and the test membrane arecompressed against one another.

The force member can apply pressure that is substantially perpendicularto the test membrane. Without being bound to any particular theory, thepressure facilitates the vertical flow. The pressure allows each layerof the membrane stack to be in contact with another layer. The pressurecan also be relieved to stop the flow so that the test sample can dwellor rest upon the test membrane, which can allow for greater sensitivity.The pressure can then be reapplied to allow the vertical flow tocontinue by allowing the sample to flow into the absorbent member(s).The force member can apply pressure such that the conjugate pad contactsa portion of the housing (e.g., first or second housing members orremovable layer). In some embodiments, the conjugate pad contacts thehousing when it is not under the pressure being exerted by the forcemember but upon the force member exerting pressure the conjugate pad iscompressed against a portion of the housing.

In some embodiments, the conjugate pad contacts the perimeter of theinlet opening. The inlet opening can also comprise a collar or othersimilar feature, such as an O-ring. In some embodiments, the conjugatepad contacts the perimeter of a collar and/or an O-ring. In someembodiments, the conjugate pad is capable of being compressed againstthe perimeter of the inlet opening, which can include, in someembodiments, a collar and/or an O-ring.

“Capable of being compressed against the perimeter of the inlet opening”refers to a membrane or pad (e.g. conjugate pad) being compressed eitherdirectly in contact with the perimeter of the inlet opening or beingcompressed against another layer or material (e.g. membrane) that is incontact with the perimeter of the inlet opening.

In some embodiments, the conjugate pad is not in direct physical contactwith the housing but is in fluid contact with the housing. “FluidContact” means that if a sample is applied to the device through theinlet opening or other opening the fluid will contact the conjugate pad.In some embodiments, the conjugate pad can be separated from the housingby another membrane, such as a permeable membrane, where the othermembrane is in direct physical contact with the housing or in directphysical contact with the collar or O-ring. When the sample is appliedto the device the fluid can contact the other membrane first and thencontact the conjugate pad. This is just one example of the conjugate padbeing in fluid contact with the housing. There are numerous otherembodiments where the conjugate pad is not in direct physical contactwith the housing, the collar, or the O-ring, but is in fluid contactwith the housing.

The force member can apply any pressure that is sufficient to facilitatevertical flow across the different membrane layers. In some embodiments,the layers of the device (e.g. conjugate pad, permeable membrane, testmembrane, and absorbent member) are compressed under a force chosen fromabout 5 lbf to 100 lbf, about 5 lbf to 50 lbf, about 10 lbf to 40 lbf,about 15 lbf to 40 lbf, about 15 lbf to 25 lbf, or about 30 lbf to 40lbf. In some embodiments, the layers of the device (e.g. conjugate pad,permeable membrane, test membrane, and absorbent member) are compressedunder a force chosen from about 1 lbf to 100 lbf, about 1 lbf to 50 lbf,about 1 lbf to 5 lbf, about 1 lbf to 10 lbf, about 1 lbf to 15 lbf,about 1 lbf to 20 lbf, about 1 lbf to 30 lbf, or about 1 lbf to 25 lbf.The force can also compress a hydrophobic or impermeable membrane aswell if one is present in the device.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the force member contacts a first surfaceof an absorbent member. In some embodiments, a conjugate pad contacts atest membrane. In some embodiments, a first surface of a test membranecontacts a permeable membrane. In some embodiments, a second surface ofthe test membrane contacts a second surface of the absorbent pad. Insome embodiments, the device comprises a hydrophobic membrane, and, forexample, the hydrophobic membrane contact a second surface of the testmembrane. In some embodiments, the hydrophobic membrane contacts a firstsurface of the absorbent pad. In some embodiments, a conjugate padcontacts an adhesive member. In some embodiments, a test membranecontacts an adhesive member.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, a first surface of the conjugate padcontacts the housing and a second surface of the conjugate pad contactsa first surface of the permeable membrane, wherein the second surface ofthe permeable membrane contacts a first surface of the test membrane,wherein a second surface of the test membrane contacts a first surfaceof the absorbent pad, wherein a second surface of the absorbent padcontacts the force member. In some embodiments, the first surface of theconjugate pad contacts a perimeter of the inlet opening of said housing.In some embodiments, the first surface of the conjugate pad contacts aperimeter of a collar or an O-ring.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, a first surface of the conjugate padcontacts the housing and a second surface of the conjugate pad contactsa first surface of the adhesive member, wherein the second surface ofthe adhesive member contacts a first surface of the test membrane,wherein a second surface of the test membrane contacts a first surfaceof the absorbent pad, wherein a second surface of the absorbent padcontacts the support member. In some embodiments, the first surface ofthe conjugate pad contacts a perimeter of the inlet. In someembodiments, the first surface of the conjugate pad contacts a perimeterof a collar or an O-ring.

The device can also comprise an attachment member. In some embodiments,the attachment member is flexible or made from a flexible material. Insome embodiments, the attachment member is fixed or made from anon-flexible material. The fixed attachment member can be, for example,a hinge and the like that can, for example, contact the conjugate pad oranother layer or membrane of the system and can mediate itsdisplacement. The fixed attachment member, such as, but not limited to,a fixed hinge or other compressible material that acts like a hinge andcan return to a shape or dimension upon compression release. Theattachment member can be capable of displacing the conjugate pad. Theattachment member can also just be plastic and although can flex, itsflexing properties are not used in the functioning of the device.

The flexible material can be, for example, an elastic or elastomermaterial. An attachment member can be, for example, attached to aconjugate pad and/or a hydrophobic membrane. The attachment member canalso be attached to any membrane or member of the device. Examples ofattachment members include, but are not limited to, elastomer band,rubber band, spring, and the like. In some embodiments, the attachmentmember can be made of a shape memory material. In some embodiments, theattachment member makes it possible to create a delay between moving thelocking member and moving the conjugate pad or any other type ofmembrane or pad that the attachment member is attached to. In someembodiments, the movement of the pad or membrane does not happen at thesame time as the sliding button or locking member is moved. In someembodiments of a device that can be used to detect multiple analyteswith a single signal, and not being bound to any particular theory, asthe sliding button or locking member is moved energy is accumulated inthe attachment member and this energy is used to pull on a pad ormembrane that it is attached to the attachment member after the pressurehas been released. In some embodiments, the locking member is moved awayfrom the force member (i.e., the force member no longer contacts thelocking member) before the conjugate pad is moved or removed. Theconjugate pad, in some embodiments, is moved once the compression orpressure being exerted by the force member is completely removed.

The attachment member can also be attached to either a sliding button orlocking member. The attachment member can be attached through any meanssuch as, adhesives, staples, tying, and the like to the othercomponents. In some embodiments, the membrane or pad has notches in themembrane or pad that allow the attachment member to attach to themembrane or pad. A non-limiting example can be seen in FIG. 9.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the housing comprises a locking member.The locking member can be a slidable locking member that can move withinthe device. The locking member can be used to lock the force member in aposition such that the force created by the force member upon thedifferent layers is maintained. The locking member is, for example,locking the force member in place so that the pressure cannot berelieved unless the locking member is moved to allow the force member tochange positions (i.e. lowered). The locking member, can for example,fit under the head of the force member, which would keep the forcemember in the raised position. The locking member can also be situatedso that it keeps the force member in a particular position (e.g. raisedor lowered). The locking member can be made of any material including,but not limited to, plastic and the like. The locking member can, forexample, contact the force member either directly or indirectly throughanother component that prevents the force member from releasing thepressure. In some embodiments, the locking member contacts the forcemember to compress the conjugate pad.

The locking member can also contact the attachment member such thatmovement of the locking member will move the attachment member, anyother membrane (e.g. conjugate pad, hydrophobic membrane, test membrane,or absorbent member) or other component that is attached to theattachment member. For example, if the locking member is moved torelieve the pressure of the force member thereby allowing the forcemember to change positions (e.g. from raised to a lower position), themovement of the locking member will also deform/accumulate energy intothe attachment member so it can move the membrane or pad once thepressure has been sufficiently reduced. When the conjugate pad isattached to the attachment member and the locking member is moved thiswill also move the conjugate pad once the pressure has been sufficientlyreduced. In some embodiments, the pressure is completely removed. Theconjugate pad can be, for example, moved such that it is removed fromthe device. In some embodiments, the conjugate pad is moved to revealthe test membrane through the inlet opening. The amount of the testmembrane that is revealed will depend upon the type of detection that isused. For a visual detection more of the test membrane may need to berevealed in the inlet opening. For a non-visual, e.g. fluorescent,near-infrared, infrared, radioactive or chemiluminescent detection, lessor none of the test membrane may need to be revealed. In someembodiments, the conjugate pad is moved so that it no longer can be seenor detected through the inlet opening. In some embodiments, the movementof the conjugate pad can create another opening other than the inletopening to visualize or detect the test membrane. In some embodiments,the conjugate pad is dissolved to visualize or detect the test membrane(e.g. detection of the analyte or multiple analytes with a singlesignal). The conjugate pad can be made of a dissolvable material suchthat when the conjugate pad comes into contact with the sample oranother solution the conjugate pad partially or completely dissolves.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the attachment member is also attached tothe impermeable or hydrophobic membrane. When the attachment member ismoved the movement will also move or remove the impermeable orhydrophobic membrane. As discussed herein, the presence of theimpermeable or hydrophobic membrane can allow the test sample to dwellor rest upon the test membrane by slowing or stopping the vertical flow.When the impermeable or hydrophobic membrane is moved or removed, eitherby its attachment to the attachment member or through other means, thevertical flow is no longer impeded or inhibited.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the housing comprises a sliding button. Asliding button can also be referred to as a sliding member. The slidingbutton can cross the inner and outer surfaces of the housing. In someembodiments, the sliding button or sliding member protrudes to an outersurface of the housing. In some embodiments, the sliding button isattached either directly or indirectly to the locking member. When thesliding button is attached (directly or indirectly) to the lockingmember the movement of the sliding button also moves the locking member.The attachment member in some embodiments can be attached to the slidingbutton. In some embodiments, the attachment member is attached to boththe sliding button and the locking member. The sliding button and thelocking member can also be constructed as a single unit.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, any one or more of the inlets comprise anopening chosen from a range of about 0.2 to about 20 cm². In someembodiments, any one or more of the inlets is about 1 to about 2 cm indiameter. In some embodiments, any one or more of the inlets is about 1or about 1.5 cm in diameter. In some embodiments, any one or more of theinlets is about 1, about 2, about 3, about 4, or about 5 cm in diameter.In some embodiments, where there is more than one inlet, the inlets canbe different sizes or the same sizes. The size of each inlet isindependent of one another. In some embodiments of the devices andsystems described herein, the devices or systems comprises 1, 2, 3, 4,or 5 inlets. In some embodiments of the devices and systems describedherein, the devices or systems comprises at least 1, 2, 3, 4, or 5inlets.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the inlet opening comprise an openingchosen from a range of about 0.2-20 cm². In some embodiments, the inletopening is about 1 to about 2 cm in diameter. In some embodiments, theinlet opening is about 1 or about 1.5 cm in diameter. In someembodiments, the inlet opening is about 1, about 2, about 3, about 4, orabout 5 cm in diameter.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, a device for detecting an analytescomprises a first member and a second member. In some embodiments, thefirst member and second member are in contact with each other. In someembodiments, the first member comprises one or more inlets. In someembodiments, between the first and second member is an analyte detectionmembrane system. In some embodiments, the analyte detection membranesystem between the first and second member comprises a conjugate pad, anadhesive member, a test membrane and an absorbent member. In someembodiments, the analyte detection membrane system comprises in thefollowing order: a conjugate pad; an adhesive member; a test membrane;and an absorbent member. As discussed herein, in some embodiments, atleast a portion of each of the conjugate pad, test membrane, andabsorbent member are substantially parallel to each other. In someembodiments, at least a portion of each of the conjugate pad, testmembrane, and absorbent member are in a different spatial plane.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the analyte detection membrane system iscompressed between the first and second member (e.g. of the forcemember). In some embodiments, the analyte detection membrane system iscompressed between a plane formed by the first member and a plane formedby the second member wherein the planes formed by the first and secondmembers are substantially parallel to each other and the analytedetection membrane system. In some embodiments, the planes are parallelto each other and the analyte detection membrane system. In someembodiments, the first and second members that compress the analytedetection membrane system is a force member. For example, the forcemember can be referred to as comprising a first and second member tocreate the force that compresses the analyte detection membrane system.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the first and second member are attachedto one another along an edge of the first member that is parallel to anedge of the second member. In some embodiments, the first and secondmember are attached by a spring, hinge, and the like. The manner bywhich the first and second member are attached is not limited and can beby any structure that enables the analyte membrane system to becompressed between the first and second member. In some embodiments, thefirst and second member are contiguous with one another and form a clip.Examples of clips (e.g. force members) are shown throughout the presentapplication (e.g. FIG. 16). The clip, can be for example cut from metalor other type of material that allows the first member to be flexiblesuch that the analyte detection membrane system can be inserted betweenthe first and second members. In some embodiments, the first member isremovable.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the first member is attached or incontact with the conjugate pad, wherein the movement or removal of thefirst member moves the conjugate pad or removes the conjugate pad fromthe device. In some embodiments, the conjugate pad is removable.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the conjugate pad is removed from thedevice comprising the first and second member by removing only theconjugate pad.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the conjugate pad comprises a tab. Thetab can be used to remove or to facilitate the removal of the conjugatepad.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the devices described herein are placedin a container. In some embodiments, the container is a pouch or a bag.In some embodiments, the container comprises an inlet. In someembodiments, the container comprises a removable or movable member orlayer that when moved or removed exposes the inlet allowing the sampleto be applied to the analyte detection membrane system. Examples of aremovable or movable member or layer includes, but is not limited to, aflap or tab. A flap or tab, for example, is shown in FIGS. 18 and 19. Insome embodiments, the removable layer or movable layer can also act as aseal for the container. The seal can protect the conjugate pad and/orthe analyte detection membrane system.

In some embodiments of the devices and systems described herein, theremovable or movable layer is in contact with or attached to theconjugate pad.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, a device for detecting an analytecomprises a first outer member and a second outer member comprising afirst inner member and a second inner member, wherein the first innermember and second inner member are in contact with each other. In someembodiments, the first outer member comprises an inlet. In someembodiments, the first inner member comprises an inlet. In someembodiments, the first outer member and the first inner member comprisean inlet. In some embodiments, between the first and second innermembers is an analyte detection membrane system. In some embodiments,the device comprises a conjugate pad. In some embodiments, the devicelacks a conjugate pad. In some embodiments, the analyte detectionmembrane system comprises a test membrane and an absorbent member andoptionally a conjugate pad. In some embodiments, the analyte detectionmembrane system comprises in the following order a test membrane and anabsorbent member. In some embodiments, at least a portion of each of theoptional conjugate pad, test membrane, and absorbent member aresubstantially parallel to each other. In some embodiments, as discussedabove, the analyte detection membrane system is compressed between thefirst inner member and second inner member. In some embodiments, thedevice and/or system comprises an adhesive member as described herein.In some embodiments, the device comprises a filtration membrane. In someembodiments, the filtration membrane can be within the analyte detectionmembrane system. In some embodiments, the a first surface of thefiltration membrane contacts a surface of the first inner member and asecond surface of the filtration membrane contacts another membrane ormember of the analyte detection membrane system. In some embodiments, asecond surface of a filtration membrane contacts a surface of a testmembrane. The filtration membrane can be any material as describedherein. For example, the filtration membrane, in some embodiments, canbe the same materials that can be a conjugate pad, test, membrane,absorbent member, and the like. In some embodiments, the filtrationmembrane is a glass fiber pad.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, where the conjugate pad is not presentwithin the device or the system, the conjugate is supplied as a liquidor as a material that can be dissolved in a liquid (e.g. water, bufferedsolution, saline, and the like). The conjugate can be supplied in aseparate container (e.g. tube) and be provided with a device or systemdescribed herein. Where the conjugate is supplied in a container theconjugate is incubated with the sample before the sample is applied tothe analyte detection membrane system. The sample can be produced by anymethod and/or as described herein. For example, a piece of meat can beswabbed or wiped and to produce a test sample. The test sample can thenbe incubated or contacted with the conjugate to produce a testsample-conjugate mixture. This mixture can then be applied to theanalyte detection membrane system as described herein using a deviceand/or system as described herein. In some embodiments, the testsample-conjugate mixture is applied directly to the test membrane. Insome embodiments, the test sample-conjugate mixture is filtered orpasses through another membrane prior to contacting the test membrane.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the analyte detection membrane system iscompressed between the first and second inner members. In someembodiments, the analyte detection membrane system is compressed betweena plane formed by the first inner member and a plane formed by thesecond inner member wherein the planes formed by the first inner memberand the second inner member are substantially parallel to each other andthe analyte detection membrane system. In some embodiments, the planesare parallel to each other and the analyte detection membrane system. Insome embodiments, the planes are substantially parallel to the first andsecond outer members.

In some embodiments of the devices described herein and throughout, theconjugate pad is not compressed by the first and second inner members orby the force members described herein.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the first outer member comprises aremovable or movable tab. In some embodiments, the conjugate pad isattached to said first outer member. In some embodiments, the conjugatepad is attached to the removable or movable tab. In some embodiments,the first outer member and second outer member form a container and thecontainer encapsulates the first and inner second member. In someembodiments, the container is a pouch, bag (e.g. sealable (e.g. zipper,adhesive, and the like) or any other type of container that canencompass the analyte detection membrane system and that is compressedbetween the first and second inner members.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the container comprises a removable ormovable tab. The removable or movable tab can be any shape and can becompletely removable or removed to an extent that exposes the inlet. Insome embodiments, the tab when moved or removed removes or moves theconjugate pad. The conjugate pad can be moved, for example, a sufficientdistance so that the results of the test membrane can be analyzed (e.g.visualized).

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, a first surface of the conjugate pad isin contact with the first outer member and a second surface of theconjugate pad is in contact with the first inner member.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the first and second inner members areattached to one another along an edge of the first inner member that isparallel to an edge of the second inner member. In some embodiments, thefirst and second inner members are attached by a spring, hinge, and thelike. The manner by which the first and second inner members areattached is not limited and can be by any structure that enables theanalyte membrane system to be compressed between the first and secondmember. In some embodiments, the first and second inner members arecontiguous with one another and form, for example, a clip. Examples ofclips are shown throughout the present application. The clip, can be forexample, cut from metal or other type of material that allows the firstinner member to be flexible such that the analyte detection membranesystem can be inserted between the first and second members. In someembodiments, the first inner member is removable.

As discussed herein, the devices and systems can comprise a removable ormovable layer (e.g. tab). The removable or movable layer can be removedor moved by manual force, such as, but not limited to, pealing ortearing. The removable or movable layer can also be removed or moved bymechanical force. The manner by which the removable or movable layer ismoved can by any means. Examples of a removable or movable layerincludes but is not limited to, tabs, flaps, and the like. As discussedherein, this flap or tab can act as a seal and the like.

As discussed herein, the conjugate pad can comprise an analyte specificcapture reagent. In some embodiments, the conjugate pad comprises aplurality of analyte specific capture reagents. In some embodiments, theconjugate pad comprises 1, 2, 3, 4, or 5 analyte specific capturereagents. The analyte can be any molecule that can be specificallyrecognized by a capture reagent. Examples of analytes include apolynucleotide molecule (e.g. DNA, RNA, siRNA, antisenseoligonucleotide) a peptide, a protein, a saccharide, a polysaccharide, acarbohydrate, and the like. The antigen can also refer to differentepitopes present on the same protein or polypeptide. The analyte canalso refer to antigens from pathogenic or non-pathogenic organisms.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the devices may be housed singly, inpairs, or in multiple configurations. The housing can be watertight toprevent leakage and can be manufactured from a variety of inertmaterials, such as polymer materials. The inlet, in some embodiments,can be of sufficient volume to contain any required amount of sample orreagents to be used with the invention.

Because the membranes, members, or pads of the device are, in someembodiments, chemically inert, they may have to be activated at anyreaction site where it is desired to immobilize a specific bindingreagent against solvent transport. Various methods may be required torender the reagent immobilized according to the particular chemicalnature of the reagent. Generally, when the media is nitrocellulose or amixed nitrocellulose ester, no special chemical linkage is required forthe immobilization of reagents. Various techniques may be used for othermaterials and reagents which include functionalization with materialssuch as carbonyldiimidazole, glutaraldehyde or succinic acid, ortreatment with materials such as cyanogen bromide. Other suitablereactions include treatment with Schiff bases and borohydride forreduction of aldehyde, carbonyl and amino groups. DNA, RNA and certainantigens may be immobilized against solvent transport by baking onto thechromatographic material. Baking may be carried out at temperaturesranging from about 60° C. to about 120° C. for times varying from aboutfive minutes to about 12 hours, and in some embodiments, at about 80° C.for about two hours.

Embodiments described herein also provide systems comprising the devicesdescribed herein and a buffer container. The systems can be used todetect multiple analytes with a single signal. The buffer container canbe any buffer that the sample that is being tested can be mixed with andthen applied to the device. For example, the sample can be taken from asource and the sample can be mixed with the buffer. The buffer can be alysis buffer that will lyse the cells or a buffer that maintains the pHof the sample so that the analysis can be done properly. The buffercontainer can be any shape and can be included outside or inside thehousing of the device.

In some embodiments, a system is provided that comprises a samplecollector. The sample collector can be any material that can take asample from a source and allow the sample to be tested. For example, thesample collector can be a swab, such as a cotton-swab. In someembodiments, the sample collector is an inoculator. In some embodiments,the housing comprises the sample collector and a portion of the samplecollector is in the inside of the housing. In some embodiments, thesample collector is partially outside and partially inside the housing.In some embodiments, the sample collector is completely outside thehousing.

Kits for detecting multiple analytes with a single signal is alsoprovided, wherein the kits comprise a device described herein. The kitcan include a device as described herein, a sample collector, a buffercontainer, an instruction manual, a positive control, a negativecontrol, or any combination thereof. With respect to the kit, a positivecontrol is a sample that is known to contain the analyte(s) that can bedetected with the device present in the kit. In contrast the negativecontrol, would not contain an analyte that can be detected by the kit.For example, the negative control when used in conjunction with theanti-antibody would be able to demonstrate that the device is workingproperly.

Buffers can also be included in the present invention. Examples ofbuffers include, but are not limited to, 1×PBS (10 mM Phosphate, 137 mMSodium Chloride, 2.7 mM Potassium Chloride), a wash buffer (e.g. 10 mMSodium Phosphate, 150 mM NaCl, 0.5% Tween-20, 0.05% Sodium Azide), amembrane buffer (e.g. 10 mM Sodium Phosphate, 0.1% Sucrose, 0.1% BSA,0.2%, PVP-40 pH 7.21, filtered with 0.2 μm filter.), PolyclonalConjugate Block Buffer (e.g. 50 mM Borate, 10% BSA, pH 8.93); PolyclonalConjugate Diluent (e.g. 50 mM Borate, 1% BSA, pH 9.09), or BlockingBuffers (e.g. 10 mM Sodium Phosphate, 0.1% Sucrose, 0.025% Silwet pH7.42; 10 mM Sodium Phosphate, 1% Sucrose, 1% Trehalose, 0.01% BSA,0.025% Tween-20; 0.05% Sodium Azide, 0.025% Silwet pH 7.4; 10 mM SodiumPhosphate, 0.1% Sucrose, 0.1% BSA, 0.2% PVP-40 pH 7.21). The buffer canalso be, but is not limited to, a blocking buffer (e.g. 10% BSA indeionized water, pH 7.4 or 1% BSA in deionized water, pH 7.4); 10 mMBorate, 3% BSA, 1% PVP40, and 0.25% Tween-100; and the like.

The conjugate pad and the test membrane can be contacted with any of thebuffers described herein either in the presence or absence of a capturereagent and, in some embodiments, allowed to dry.

Examples of buffers that are lysis buffers include, for example, but arenot limited to, 2% Tween (v/v) and 0.1% Triton(v/v); 2% Tween(v/v) and0.1% SDS(w/v); 2% Tween(v/v) and 0.1% BSA(w/v); 2% Tween(v/v) and 1%BSA(w/v), 0.1% SDS(w/v), 1% BSA(w/v), or any combination thereof. Thelysis buffers can also be, for example, 5% Tween/PBS; 2% Tween/PBS+0.1%SDS; 2% Tween/PBS+1% BSA. Other examples of lysis buffers include, butare not limited to, 5% Tween-80 (v/v); 5% Triton X-100 (v/v); 5% NP40(v/v); 2% Tween-80 (v/v); 2% Triton X-100 (v/v); 2% NP40 (v/v); 1%Tween-80 (v/v); 1% Triton X-100 (v/v); and 1% NP40 (v/v). The detergentsand other components of the buffers can be made with any suitable buffersuitable for proteins, and includes, but is not limited to, water andphosphate buffered saline. The lysis buffers can be used to prepare thesamples prior to the samples making contact with the devices describedherein. In some embodiments, a lysis buffer is not used. A lysis bufferis not used on a sample when a surface protein or surface analyte isdesired to be detected in the method. Accordingly, in some embodiments,the sample is not subject to lysis or conditions that would cause a cellto be lysed. Where a cell is being used the cell could be part of thebridging complex and replace an amplicon that is shown, for example, inFIG. 3. The cell could be labeled or unlabeled so long as there is acapture reagent that can create a similar bridging complex.

The present subject matter also provides for methods of detectingmultiple analytes comprising contacting a sample with a device and/orsystem as described herein, wherein the sample contacts the conjugatepad and the test membrane, wherein a positive reaction with the testmembrane indicates the presence of the multiple analytes. In someembodiments, the conjugate pad comprises a signal detection unit or acapture regent that binds to the signal detection unit. In someembodiments, the test membrane comprises a second analyte-specificcapture reagent. This can bind to an interaction unit present on theanalyte. The sample can have, for example, the differentially labeledamplicons. For example, the test membrane can comprise a first capturereagent that binds to an interaction unit present on the first analyte.The conjugate pad can have a capture reagent that binds to the signaldetection unit. The analytes can be incubated with the bridging unitand/or the signal detection unit prior to being applied to the deviceand contacting the conjugate pad and/or test membrane. A positivereaction is indicated when the complex of the analytes, capturereagents, and signal detection units are present. Otherwise the signalis not generated. A capture reagent can be applied to the test membraneso that it will indicate a positive reaction when it binds to itsspecific binding partner. The system and devices can be utilized to formthe complexes described herein. For example, after a PCR reaction occursthat creates differentially labeled amplification products, the productsare incubated with the antibodies that can be used to create thebridging complex. The incubation mixture is then added to the device.The sample flows through the conjugate pad that contains a capturereagent and then interacts with the test membrane that contains anothercapture reagent. In some embodiments, the conjugate pad is removed ormoved so that the signal can be detected. Movement of the conjugate padin a vertical flow device is described herein. If all of the analytesare present, the bridging complex is created and the single signal canbe detected. The specific capture reagent on the test membrane can beapplied in any manner such that when it is detected it can form a line,a circle, a plus sign, a broken line, an “X” or any other pattern. Insome embodiments, the control line indicating that the device is workingproperly will cross the analyte specific line and when the multipleanalytes are present and detected the detectable label will form a plussign. The detection can be determined by the detection of the detectionreagent as described herein and by routine methods known to one of skillin the art. Similar methods can be used, for example, in an ELISAsystem.

In some embodiments, a sample contacts the device, which is thenfollowed by a buffer being applied to the device after the sample hascontacted the device. For example, a sample comprising the analytes canbe contacted with the conjugate pad such that the sample is transferredto the conjugate pad. Following the contact with the conjugate pad aseparate solution can be applied to the device to facilitate or initiatethe vertical flow through the devices described herein.

In some embodiments, as described herein, the capture reagent is anantibody. In some embodiments, the sample that is tested is a solutionbut can also be a mixture of solution or buffer and solid material thatcan be applied to the device. The solution will then solubilize theanalyte(s) and allow the conjugate pad's capture reagent to come intocontact with the appropriate analyte present in the sample. In someembodiments, the sample comprises a cell lysate. In some embodiments,the cell lysate has been clarified by centrifugation or other means toremove non-soluble materials.

In some embodiments, the methods comprise contacting a test sample witha sample collector and contacting the sample collector with the device.The test sample can be a sample comprising amplicons which are createdfrom multiple analytes. In some embodiments, the methods comprisecontacting the sample collector with a solution or buffer, wherein thesolution or buffer is applied to the device. In some embodiments, thesamples are contacted with the conjugate pad prior to the sample cominginto contact with the test membrane. In some embodiments, the sample iscontacted with the conjugate pad and the test membrane simultaneously.

In some embodiments, the method comprises moving the conjugate pad ofthe devices described herein, wherein the movement of the devicesexposes the test membrane for detection. In some embodiments, thelocking member moves the conjugate pad. In some embodiments, theconjugate pad is attached to the locking member and/or the slidingbutton member. In some embodiments, movement or removal of the removablemember moves or removes the conjugate pad. In some embodiments, theconjugate pad is attached to the removable member and/or the adhesivemember. In some embodiments, when the removable member is moved orremoved the adhesive member is also moved with respect to its originalposition or removed from the device. The analyte can be those that arediscussed herein or any other analyte that can be detected using themethods and devices described herein. In some embodiments, the methodcomprises applying the sample to the device and allowing the sample toflow through the device via vertical flow.

In some embodiments the detection or indication of the presence orabsence of multiple analytes occurs in less than 60 seconds. In someembodiments, the detection or indication of the presence or absence ofmultiple analytes occurs in about 30 to about 60 seconds. In someembodiments, the detection or indication of the presence or absence ofmultiple analytes occurs in less than 2 minutes. In some embodiments,the detection or indication of the presence or absence of multipleanalytes occurs in about 30 seconds.

In some embodiments, devices for detecting multiple analytes with asingle signal are provided. In some embodiments, the device comprises ahousing. The device can comprise a first housing member and a secondhousing member to form the housing. In some embodiments, the first andsecond housing members are separate members. The first and secondhousing members can be manufactured as a single piece. The single piece,in some embodiments, can be separated into the two housing members toallow for the introduction of the materials into the housing (e.g.device). In some embodiments, the device comprises an inlet. The inletcan be in either housing member (e.g. first or second housing member).The inlet can be oriented above the conjugate pad, such that a samplethat is introduced into the device through the inlet contacts theconjugate pad prior to contacting the test membrane. The device isoriented such that regardless of any pressure being applied to thedevice, the sample will flow vertically down through the layers ofmembranes (e.g. analyte detection membrane system). Accordingly, in someembodiments, the second housing member comprises the inlet opening. Insome embodiments, the second housing member is on top of the firsthousing member. The inlet can be any size or shape as described hereinso long as the size and shape is sufficient for the introduction of asample into the device such that the sample can contact the analytedetection membrane system.

The device can comprise one or more force members. The force members canapply pressure to the analyte detection membrane system. The force isapplied perpendicular or substantially perpendicular to the membranes orlayers of the analyte detection membrane system. In some embodiments,the device comprises at least 1, 2, 3, 4, or 5 force members. In someembodiments, the device comprises at least 1, 2, 3, 4, or 5 forcemembers. In some embodiments, the device comprises a plurality of forcemembers. The force members can be in contact with a housing member. Insome embodiments, a first surface of the force member is in contact witha housing member (e.g. first or second housing member). In someembodiments, a second surface of the force member contacts the analytedetection membrane system. As described herein, the force member can beused to compress the analyte detection membrane system to facilitate theflow of the sample through the analyte detection membrane system. Thepressure can facilitate the sample to flow vertically through theanalyte detection membrane system. The force members can be oriented inthe device independently of one another. The force members can also bemanipulated such that each force member applies a pressure to a distinctanalyte detection membrane system and that the force applied to eachanalyte detection membrane system is different or, in some embodiments,the same or substantially the same.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the device comprises one or more movablelocking members. In some embodiments, the movable locking membercontacts a force member. In some embodiments, the movable locking membercontacts each force member present in the device. For example, in adevice comprising a first and second force members, the movable lockingmember is in contact with the first force member and the second forcemember. The movable locking member, in some embodiments, supports theforce member such that the force member is in a raised position. Theraised position can be determined by comparing the force member'sposition when it is in contact with the movable locking member to whenthe force member is not in contact with the movable locking member. Inthe absence of contact between the force member and the movable lockingmember, the force member is in a first position. When the movablelocking member is in contact with the force member, the force member isin a second position. In some embodiments, the second position of theforce member is considered to be a raised position. The raised positioncan be used to compress the layers (e.g. membranes) of the analytedetection membrane system. When the movable locking member is not incontact with the force member, in some embodiments, the analytedetection membrane system is not compressed.

The device can comprise one or more movable locking members. In someembodiments, the device comprises a plurality of, or 1, 2, 3, 4, or 5movable locking members. In some embodiments, the device comprises atleast 1, 2, 3, 4, or 5 movable locking members. In some embodiments, thedevice comprises a number of movable locking members that is equal tothe number of force members present in the device.

The movable locking members can also comprise a moving member, such as,but not limited to, a handle. The moving member can be used, forexample, to turn or move the movable locking member such that thelocking member contacts the force member. In some embodiments, themoving member disengages the locking members from the force member suchthat the force member changes positions (e.g. from a raised position toa lower position). The moving member can be used to relieve or apply thepressure being applied on the analyte detection membrane system. Themoving member can also be used to relieve or apply compression of theanalyte detection membrane system. In some embodiments, the movingmember rotates the locking member around a central axis of the device.For example, after applying the sample to the device and the sampleflows through at least one analyte detection membrane system, the movingmember is moved, which rotates the movable locking member in either aclockwise or counterclockwise direction. The rotation of the movablelocking member allows the force member to be lowered into a differentposition. The rotation of the movable locking member can also allow thepressure that is applied to the analyte detection membrane system to berelieved. In some embodiments, the pressure is completely relieved, or,in some embodiments, the pressure is only partially relieved.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the moving member that moves the movablelocking member protrudes through the first or second housing member. Insome embodiments, the moving member is accessible through the movingmember outlet. In some embodiments, the moving member rotates around acentral axis of the device when moved. In some embodiments, the movingmember moves the movable locking member laterally (e.g. horizontally) orvertically. In some embodiments, the movable locking member moveslaterally (e.g. horizontally) or vertically when moved.

The moving member and the movable locking member can be constructed as asingle piece or as two pieces. In some embodiments, where the movablelocking member and the moving member are two separate pieces and areconstructed to interact with one another such that the movement of onemoves the other. For example, one of the two pieces can have a “malemember” that protrudes from the surface and inserts into the “femalemember” of the other piece to form the interaction.

The movement of the movable locking member by the moving member can alsobe used to move or remove the conjugate pad present in the analytedetection membrane system. As discussed herein, the conjugate pad can beremoved to allow visualization or the analysis of the test membrane. Theconjugate pad, as discussed herein, can be removed completely from theanalyte detection membrane system or an amount that is sufficient toallow visualization or analysis of the test membrane. Analysis of thetest membrane can be based solely upon visual inspection, or in someembodiments, an optical reader can be used to analyze the test membraneto determine the absence or presence of an antigen in the sample.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the device comprises a plurality, or twoor more analyte detection membrane systems. In some embodiments, thedevice comprises at least 1, 2, 3, 4, or 5 analyte detection membranesystems. In some embodiments, the device comprises 1, 2, 3, 4, or 5analyte detection membrane systems. The analyte detection membranesystem can be as described herein and throughout the presentapplication.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the device comprises one or more flexibleor non-flexible attachment members. In some embodiments, the devicecomprises a plurality of flexible or non-flexible attachment members. Insome embodiments, the device comprises at least 1, 2, 3, 4, or 5flexible or non-flexible attachment members. In some embodiments, thedevice comprises 1, 2, 3, 4, or 5 flexible or non-flexible attachmentmembers. In some embodiments, the flexible or non-flexible attachmentmember contact the movable locking member. In some embodiments, theflexible or non-flexible attachment member contact the movable lockingmember and the conjugate pad. The flexible or non-flexible attachmentmember can be used to remove or move the conjugate pad away from therest of the layers (e.g. membranes) of the analyte detection membranesystem. In some embodiments, the device comprises a number of flexibleor non-flexible attachment members that is equal to the number ofanalyte detection membrane systems present in the device. In someembodiments, the device comprises a number of flexible attachmentmembers that is equal to the number of force members present in thedevice. The flexible or non-flexible attachment members can also be usedto retract the conjugate pad so as to reveal or expose a portion or allof the test membrane.

For example, in some embodiments, a device comprises three analytedetection membrane systems and three force members. A device with morethan one analyte detection membrane system can be used to detectdifferent analytes or different multiple analyte sets. In such a device,for example, the device comprises a first, second, and third attachmentmember. The first attachment member can be in contact with the conjugatepad of the first analyte detection membrane system and a movable lockingmember. Additionally, in some embodiments, the second attachment membercan be in contact with the conjugate pad of the second analyte detectionmembrane system and a movable locking member. In some embodiments, thethird attachment member can be in contact with the conjugate pad of thethird analyte detection membrane system and a movable locking member. Insome embodiments, the first, second, and third attachment members are incontact with the same movable locking member. In some embodiments, thefirst, second, and third attachment members are in contact withdifferent movable locking members. For example, in some embodiments, thefirst and second attachment members are in contact with the same movablelocking member and the third attachment member is in contact with adifferent movable locking member. Each attachment member isindependently contacted with one or more movable locking members.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the movable locking member comprises oneor more movable locking member extensions. In some embodiments, themovable locking member extensions contacts a force member. In someembodiments, the device comprises a number of movable locking memberextension that is the same as the number of force members that arepresent in the device. In some embodiments, the movable locking memberextension partially encircles or encompasses the force member. In someembodiments, the movable locking member extension completely encirclesor encompasses the force member. The shape of the movable locking memberor member extension can be any shape to keep the force member in araised position. In some embodiments, the extension is a hook orhook-like shape that partially or completely encircles or encompassesthe force member. The shape is not essential so long as the shape actsas a support for the force actuator (e.g. force member).

The number of movable locking member extensions can the same ordifferent as the number of force members present in a device describedherein. In some embodiments, a device comprises a plurality of movablelocking member extensions. In some embodiments, a device comprises atleast 1, 2, 3, 4 or 5 movable locking member extensions. In someembodiments, a device comprises 1, 2, 3, 4 or 5 movable locking memberextensions. For example, in some embodiments, a device comprises afirst, second, and third force members attachment members and a first,second, and third movable locking member extensions. In thisnon-limiting example, for example, the first force member contacts thefirst movable locking member extension, the second force member contactsthe second movable locking member extension, and the third force membercontacts the third movable locking member extension.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the movable locking member comprises anattachment member extension, which can be flexible or inflexible. Insome embodiments, the attachment member extension contacts theattachment member. In some embodiments, the attachment member extensioncomprises an attachment member extension nodule. The nodule can be anyshape or size that allows the attachment member to be secured to so thatthe attachment member securely maintains its contact with the movablelocking member. In some embodiments, the one or more movable lockingmember extensions extend radially (e.g. outward) from the center of themovable locking member.

The number of attachment member extension can the same or different asthe number of analyte detection membrane systems present in a devicedescribed herein. In some embodiments, a device comprises a plurality offlexible or non-flexible attachment member extensions. In someembodiments, a device comprises at least 1, 2, 3, 4 or 5 flexible ornon-flexible attachment member extensions. In some embodiments, a devicecomprises 1, 2, 3, 4 or 5 flexible or non-flexible attachment memberextensions. For example, in some embodiments, a device comprises afirst, second, and third attachment members and a first, second, andthird attachment member extensions. In this non-limiting example, forexample, the first attachment member contacts the first attachmentmember extension, the second attachment member contacts the secondattachment member extension, and the third attachment member contactsthe third attachment member extension.

In some embodiments, the devices described herein comprise flexible andnon-flexible attachment members and/or member extensions. Throughout thepresent disclosure, reference made be made to an attachment member ormember extensions that are flexible or non-flexible. If one embodimentdiscloses a flexible member it is understood that another embodiment isalso disclosed where the member is non-flexible unless context dictatesto the contrary.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the device comprises a channel system.The channel system can be used to transport the sample (e.g. fluid) fromthe inlet opening of the device to the analyte detection membranesystem(s) present in the device. As used herein, the “channel system”refers to the entire system regardless of how many individual channelsare a part of the system. For example, the channel system can comprisestwo or more channels, such as, but not limited to, capillaries, thattransport fluid from the inlet to an analyte detection membrane system.In some embodiments, the channel system comprises one or more branches(e.g. arms). The one or more branches can be transport fluid to one ormore analyte detection membrane systems. In some embodiments, thechannel system comprises 1, 2, 3, 4, or 5 branches. In some embodiments,the device comprises a number of branches in the channel system that isequal to the number of analyte detection membrane systems present in thedevice.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, each branch of the channel systemcomprises capillary tubes that transport the fluid from the inlet to theanalyte detection membrane system. In some embodiments, each branchcomprises a plurality of capillary tubes. In some embodiments, eachbranch comprises at least 1, 2, 3, 4, or 5 capillary tubes. In someembodiments, the channel system does not comprise capillary tubes ortube-like formations but is made from a material that allows a portionof the sample to be transported from the inlet to the conjugate pad ofthe analyte detection system. In some embodiments, the channel system isa porous material that can be used to transport the sample from theinlet to the analyte detection membrane system. In some embodiments, thechannel system is made from the same material as the conjugate pad. Insome embodiments, the channel system and the conjugate pad are acontiguous piece of material. In some embodiments, the channel systemcomprises a Porex material. These porous materials allow the inlet to bein fluid communication with the analyte detection membrane system. Insome embodiments, the porous material comprises polyethylene,polypropylene, polytetrafluourouethylene (PTFE), PVDF, ethyl vinylacetate, Nylon 6, thermoplastic polyurethane (TPU), SCP, and the like.In some embodiments, the conjugate pad and the channel system are thesame materials or different materials. In some embodiments, the channelsystem does not comprise a porous material and/or a capillary tubesystem.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the channel system contacts the inlet. Insome embodiments, the channel system contacts the top of the analytedetection membrane system. In some embodiments, the channel systemcontacts the top of the conjugate pad or a membrane that is on top ofthe conjugate pad. In some embodiments, the channel system contacts anedge of the conjugate pad or an edge of a membrane that is on top of theconjugate pad. Regardless of how the sample contacts the analytedetection membrane system, in some embodiments, the sample flowsvertically through analyte detection membrane system. Therefore,although the sample may flow horizontally (e.g. laterally) from theinlet to the analyte detection membrane system, the sample is notanalyzed until it flows vertically through the analyte detectionmembrane system. This is distinctly different from lateral flow systemswhere a sample flows laterally (e.g. horizontally) through multiplemembranes or test layers.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the channel system divides the sampleinto equal portions, wherein each equal portion contacts an independentanalyte detection membrane system. In some embodiments, the channelsystem divides the sample into one or more unequal portions. The one ormore unequal portions are then transported to independent analytedetection membrane systems.

For example, in a device that comprises a first and second analytedetection membrane systems the device comprises a channel system thatcomprises a first and second branch. In some embodiments, the firstbranch contacts the first analyte detection membrane system and thesecond branch contacts the second analyte detection membrane system.Upon application of the sample to the device (e.g. through the inletopening), the sample is transported in equal portions through the firstand second branches of the channel system to the first and secondanalyte detection membrane systems. In some embodiments, the sample istransported in unequal portions through the first and second branches ofthe channel system to the first and second analyte detection membranesystems. The sample can be divided into unequal portions, for example,based upon the number of capillaries present in each branch. Forexample, the first branch can comprise more capillaries than the secondbranch. The greater number of capillaries will allow more of the sampleto be transported through the first branch than the second branch,thereby delivering unequal portions to the first and second analytedetection membrane systems.

Accordingly, the branches of the channel system may have the same numberof capillaries or different numbers of capillaries. The numbers ofcapillaries in each branch of the channel system is independent of eachbranch. That is each branch of the channel system can have the samenumber or a different number of capillaries as another branch.Therefore, in some embodiments, the device's channel system can bedescribed as a capillary channel system. In some embodiments, thechannel system is enclosed in a channel housing that is separate anddistinct from the first and second housing members described herein forthe device itself. In some embodiments, the channel housing istransparent, translucent, opaque, or partially translucent.

As discussed herein, the test membrane can be analyzed either visuallywith the human eye or through a machine, such as an optical reader todetermine the presence or absence of multiple analytes with a singlesignal. In some embodiments, the analysis is done through a portal. Insome embodiments, the device comprises one or more portals that aresufficient in size to allow visualization of a test membrane of one ormore of the analyte detection membrane systems. In some embodiments, asingle portal is used to visualize each of the test membranes present inthe device. In some embodiments, the device does not comprise a portal.In embodiments, where the device does not comprise a portal, the testmembrane can still be visualized by using a transparent or translucenthousing for the device. In some embodiments, the first and/or secondhousing are transparent or translucent. Where the first and/or secondhousings are transparent or translucent this can allow an analytedetection membrane systems and its test membrane when it is revealedupon moving or removing the conjugate pad. In some embodiments, thedevice comprises a plurality of portals. In some embodiments, the devicecomprises at least 1, 2, 3, 4, or 5 portals. In some embodiments, thedevice comprises 1, 2, 3, 4, or 5 portals. In some embodiments, a devicecomprises 1 portal that is continuous and exposes each analyte detectionmembrane system present in the device to visual inspection.

As discussed herein, the force members can be allowed to move between atleast two positions (e.g. raised or lowered; engaged or disengaged). Insome embodiments, the force member is lowered and is encompassed by aforce actuator outlet. Thus, in some embodiments, the device comprisesone or more force actuator outlets that that can accept the force memberas it is lowered. In some embodiments, the device comprises a pluralityof force actuator outlets. In some embodiments, the force actuatoroutlet is a groove. In some embodiments, the force actuator outlet is acircle or substantially circular. The force actuator outlet can be usedto suspend the force actuator (e.g. force member) at a particularposition. The force actuator outlet can also be used to retain the forceactuator in a second position. In some embodiments, the circumference ofthe force actuator outlet is greater than the circumference of theportion of the force member that is entering the outlet. In someembodiments, the circumference of the force actuator outlet is greaterthan the largest circumference of the force member. In some embodiments,the circumference of the force actuator outlet is not greater than thelargest circumference of the force member, wherein the force member hasareas with at least two different circumferences. For example, forcemembers are described herein that would have two differentcircumferences. A force member can comprise a cap with one circumferenceand a support structure that supports the cap with a differentcircumference. The support structure can, in some embodiments, have asmaller circumference than the cap. In some embodiments, the forceactuator outlet can have a circumference that is larger than the supportstructure circumference, but smaller than the cap structurecircumference. In some embodiments, the number of force actuator outletsis the same or different than the number of the force members present ina device.

The force actuator outlet can also be a continuous depression in ahousing member that can accept each and every force member in the devicewhen it is lowered and no longer compressing the analyte detectionmembrane system. The outlet can be used to temporarily house the forcemember or it can be permanent, such that the force member cannot beraised again to compress or further compress the analyte detectionmembrane system.

As discussed herein and throughout the present application, theconjugate pad, permeable membrane, test membrane, and absorbent membercan be or are compressed by the force member under certain forces asdescribed herein and including, but not limited to a force from about 1lbf to about 100 lbf. In some embodiments, where there are multipleanalyte detection membrane systems, the pressure applied to eachmembrane detection system can be different or it can be the same. Forexample, in a device that has a first, second, and third analytedetection membrane system, the first analyte detection membrane systemcan be compressed under a force of 5 lbf, the second analyte detectionmembrane system can be compressed under a force of 10 lbf, and the thirdanalyte detection membrane system can be compressed under a force of 25lbf. In another example, in some embodiments, the first and secondanalyte detection membrane systems are compressed under the samepressure and the third analyte detection membrane system is compressedunder a pressure that is different from the first and second analytedetection membrane systems. The differences in pressure can be used touse different flow rates, which can be useful for different analytes.The pressure is correlated with the flow rate. The pressure can bemanipulated by the position of the force member and how much the layersof the analyte detection membrane system are compressed. The specificforce used can be determined and measured by one of skill in the artusing known and routine methods.

As described herein, in some embodiments, the present invention providesa system comprising any device described herein, a buffer containerand/or a sample collector. In some embodiments, the present inventionprovides a kit comprising any device described herein and one or more ofa positive control, a negative control, an instruction booklet, a buffercontainer, and/or a sample collector, or any combination thereof.

The methods described herein can be used with a device that has, forexample, a plurality, two or more, analyte detection membrane systems.The methods can be also be used with devices that have 2, 3, 4, or 5analyte detection membrane systems. Where there are more than twoanalyte detection membrane systems (e.g. 3, 4, 5, 6, 7, 8, 9, or 10) themethods and the descriptions contained herein are modified to beconsistent with the number of analyte detection membrane systems. Thesechanges are made in accordance with the descriptions contained hereinand any routine changes that would be known by one of skill in the art.The changes to encompass more than 2 analyte membrane detections systemsbased upon the descriptions contained herein combined with knowledge ofone of skill in the art would not require undue experimentation. In someembodiments as described herein, the device comprises two or moreanalyte detection membrane systems. In some embodiments, the methodcomprises contacting a sample (e.g. the sample comprising multipleanalytes) with the device and a portion of the sample being transportedthrough a channel system to the conjugate pads of the two or moreanalyte detection membrane systems. In some embodiments, the methodcomprises detecting a positive or negative reaction for the analytes,wherein a positive reaction indicates that the presence of the multipleanalytes. In some embodiments, the two or more analyte detectionmembrane systems are compressed by the force member. In someembodiments, the sample vertically flows from the conjugate pad to thetest membrane. In some embodiments, the method further comprisescompressing the analyte detection membrane system by the force member.In some embodiments, the method comprises moving the conjugate pad ofthe two or more detection systems after a portion of the sample hascontacted and flowed through the conjugate pad, thereby exposing thetest membrane for analysis. In some embodiments, the test membrane isexposed within the portal opening for detection. In some embodiments,the conjugate pad of the two or more detection systems is moved bymoving the movable locking member. In some embodiments, the moving themovable locking member comprises rotating the movable locking memberaround the central axis of the device. In some embodiments, the movablelocking member is moved laterally or vertically. In some embodiments,the moving lockable member moves the conjugate pad of the two or moredetection systems simultaneously or sequentially. In some embodiments,the method further comprises relieving the compression of the two ormore analyte detection systems. The pressure can be relieved orlessened, for example, by moving the movable locking member. In someembodiments, the movable locking member is moved (e.g. rotated) byturning or moving the moving member that is connected to the movablelocking member.

In some embodiments, one or more of the analyte detection membranesystems are compressed prior to the sample contacting the channelsystem. In some embodiments, one or more of the analyte detectionmembrane systems are compressed prior to the sample coming into contactwith the conjugate pad of the one or more of the analyte detectionmembrane systems. In some embodiments, each of the analyte detectionmembrane systems is compressed simultaneously. In some embodiments, eachof the analyte detection membrane systems is compressed independently.In some embodiments, each of the analyte detection membrane systemspresent in a device is compressed prior to a sample coming into contactwith the conjugate pad.

In some embodiments, the method comprises relieving the pressure appliedby a force member on the two or more analyte detection membrane systems,wherein said force member moves vertically or horizontally to relievesaid pressure. In some embodiments, the method comprises the forcemember moving from a first position to a second position to relieve thepressure. In some embodiments, the force member moves into or comes intocontact with a force actuator outlet when the movement of the forcemember relieves or reduces the pressure or relieves or reduces the forcebeing applied to the analyte detection membrane system. In someembodiments, the force member drops partially or completely out of thedevice.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the present invention provides a devicefor detecting an analyte comprising a pressure actuator, a pressurerelease, an analyte detection membrane system, an analyte detectionmembrane system receptacle, and an outlet. In some embodiments, theanalyte detection membrane system receptacle is of sufficient size andshape to accept the analyte detection membrane system. In someembodiments, the receptacle is a groove. In some embodiments, thereceptacle is a case that can be, but not necessarily, removed from thedevice.

In some embodiments, the analyte detection membrane system, as describedherein, can be encompassed or contained within a cartridge or housing.The housing can comprise a first and/or second housing member. In someembodiments, where the analyte detection membrane system is containedwithin a housing or a cartridge, the receptacle is of sufficient sizeand shape to accept the housing or the cartridge. In some embodiments,the housing or cartridge comprises an inlet. The inlet can be used toapply the sample to the analyte detection membrane system. In someembodiments, the cartridge or housing comprises a second outlet thatallows the sample to flow through and out of the housing and cartridge.The analyte detection membrane system can be any analyte detectionmembrane system as described herein.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the device comprises a pressure actuator.The pressure actuator, for example, can be the force member that isdescribed in herein. In some embodiments, the pressure actuator is anair valve or vacuum valve that either applies air pressure to theanalyte detection membrane system or draws a vacuum through the analytedetection membrane system. In some embodiments, the pressure actuatorcan be regulated by a pressure release or pressure regulator. Thepressure release or pressure regulator can be, for example, a vacuumrelease. The release or regulator can be used to regulate the pressureor vacuum being applied to the analyte detection membrane system. Thepressure or vacuum can be applied to the analyte detection membranesystem through an outlet or tube that is present in the device. Theoutlet can be the same outlet present in the cartridge or housingdescribed herein or it can be a different outlet or tube. The outlet ortube can be used so that the pressure or vacuum to be applied withspecificity rather than allowing it to diffuse across the entire device.

In some embodiments, the housing (e.g. cartridge) encasing the analytemembrane detection comprises an upper housing and a lower housing. Insome embodiments, the housing comprises a plurality of membrane or padholders. In some embodiments, the housing comprises one or more membraneor pad holders. In some embodiments, the housing comprises 1, 2, 3, 4,or 5 membrane or pad holders. In some embodiments, the housing comprisesat least 1, 2, 3, 4, or 5 membrane or pad holders. In some embodiments,the housing comprises an inlet. In some embodiments, the housingcomprises an outlet. In some embodiments, the vacuum actuator directlyor indirectly contacts the housing outlet.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the device and any device describedherein comprises an ejector for ejection the housing. The ejector can beused to facilitate the removal of the housing that contains the analytedetection membrane system. In some embodiments, the devices comprise ahousing separator. The housing separator can be used to facilitate theseparation of the housing. In some embodiments, the ejector can also actas the housing separator.

In addition to the methods described herein, in some embodiments, amethod of detecting multiple analytes comprises applying a samplecontaining the multiple analytes to a device comprising a pressureactuator, a pressure regulator, an analyte detection membrane system, ananalyte detection membrane system receptacle, and an outlet or any otherdevice or analyte detection membrane system described herein. In someembodiments, the sample is contacted with the analyte detection membranesystem, wherein the sample vertically flows through the analytedetection membrane system. In some embodiments, the method comprisesdetecting the presence or absence of the analyte. This can be doneaccording to the bridging complex that is formed through the use of theinteraction units, capture reagents, and signal detection unitsdescribed herein.

In some embodiments of using the devices to detect multiple analytes,detecting comprises removing or moving the conjugate pad present in theanalyte detection membrane system a sufficient amount to visualize theresult, wherein a positive result indicates the presence of the multipleanalytes. In some embodiments, detection comprises removing the analytedetection membrane system from the device and further removing or movingthe conjugate pad a sufficient amount to visualize the detection of theanalyte or multiple analytes with a single signal. In some embodiments,the analyte detection membrane system is contained within a housing orcartridge, and therefore, in some embodiments, the housing or cartridgeis removed from the device prior to the movement or removal of theconjugate pad. In some embodiments, the housing is separated into afirst (e.g. upper) and a second (e.g. lower) housing prior to theremoval or movement of the conjugate pad as described herein. In someembodiments, the separation of the housing into a first and a secondhousing removes or moves the conjugate pad to visualize the testmembrane as described herein. In some embodiments, the housing isseparated manually and/or mechanically. In some embodiments, the housing(e.g. cartridge) is ejected from the device. In some embodiments, thehousing is ejected from the device by an ejector. In some embodiments,the housing is separated by a separator. In some embodiments, theejector also functions as a separator.

In some embodiments, the method comprises applying pressure on ordrawing a vacuum through an analyte detection membrane system. In someembodiments, the method comprises releasing or reducing the pressure orthe vacuum. In some embodiments, the pressure or vacuum is released orreduced by using the pressure regulator. In some embodiments of themethods described herein, the sample that is contacted with the analytedetection membrane system flows through the analyte membrane system at aflow rate that is regulated by a pressure actuator. In some embodiments,the entire sample flows through the analyte detection membrane system ata constant rate. In some embodiments, the sample flows through theanalyte detection membrane system at a variable rate. In someembodiments, the variable rate comprises at least one period of timewhere the flow rate of a portion of the sample is 0. For example, thepressure being applied or vacuum being drawn can be regulated such thatthe sample stops flowing through the analyte detection membrane systemfor a period of time. This can be referred to as a “dwell.” As describedelsewhere in the present document, the dwell can be implemented byplacing impermeable or slightly impermeable membranes between theconjugate pad and the other layers of the analyte detection membranesystem. The dwell, however, can also be regulated by regulating (e.g.changing) the pressure that is applied to the analyte detection membranesystem. The dwell can also be regulated by regulating (e.g. changing)the vacuum that is being drawn through the analyte detection membranesystem. Any method of regulating the flow rate through the analytedetection membrane system, including but not limited to, the flow ratethrough the conjugate pad and/or the test membrane can be used.

The devices herein, can also be automated or used in conjunction with anoptical reader or other type of spectrometer. The advantages ofcombining the systems and devices described herein with an opticalreader or other type of spectrometer is that the sensitivity of thedevices and assays can be increased such that less analyte present inthe sample is necessary to identify a sample as being positive for thatanalyte. This greater sensitivity can be then be used, for example, todetermine if food contains pathogens, a person has a certain disease orcondition, or if a product has an analyte that is otherwise undetectableusing other devices and methods in the same amount of time it takes touse the presently described methods and devices.

Accordingly, in some embodiments of a device that can be used to detectmultiple analytes with a single signal, the present invention provides adevice for detecting multiple analytes comprising a sample inlet, ananalyte detection cartridge receptacle, an analyte detection cartridgereceptacle inlet, an optional conjugate pad remover, a pressureactuator, a spectrometer (e.g. optical reader), a display unit, a signalprocessing unit. The pressure actuator can be a force member whoseposition is modified to regulate the pressure being applied to theanalyte detection membrane system that is used in conjunction with adevice. The pressure actuator can also regulate the pressure by drawinga vacuum through the analyte detection membrane system that is used inconjunction with a device. The spectrometer can be any spectrometer thatcan detect the presence of a signal. The signal can be an opticalsignal. The signal can be a signal that is emitted in a spectrum chosenfrom, for example, infrared spectrum; near-infrared spectrum; visiblespectrum, x-ray spectrum, ultra-violet spectrum, gamma rays,electromagnetic spectrum, and the like.

The spectrometer can be connected to the signal processing unit (e.g.computer). The signal processing unit can take the signal that istransmitted to it and analyze the signal to determine the presence orabsence of the sample. An example of a signal processing unit is, butnot limited to, a computer. The signal processing unit can programmed toanalyze the signal transmitted by the spectrometer. The programming canimplement an algorithm to analyze the signal to determine the presenceor absence of an analyte in the sample. The algorithm can be based uponcriteria that are pre-installed in the signal processing unit's memoryor that are entered by the user of the device. The types of informationthat can be entered can be cut-offs for a positive or negative signal,processing times, and the like. The signal processing unit can also beused to regulate the pressure applied to or the vacuum drawn through theanalyte detection membrane system.

The signal processing unit can be used or programmed to regulate theflow rate of the sample through the analyte detection membrane system.The flow rate can be regulated by regulating the pressure that isapplied to or a vacuum that is drawn through the analyte detectionmembrane system. As described above with respect to the methodsdescribed herein, the sample that is contacted with the analytedetection membrane system flows through the analyte membrane system at aflow rate that is regulated by a pressure actuator. The pressureregulator can be in turn regulated by, for example, the signalprocessing unit. In some embodiments, the entire sample flows throughthe analyte detection membrane system at a constant rate, which isregulated by the signal processing unit. In some embodiments, the sampleflows through the analyte detection membrane system at a variable rate,which is regulated by the signal processing unit. In some embodiments,the variable rate comprises at least one period of time where the flowrate of a portion of the sample is 0, which can be regulated by thesignal processing unit. For example, the pressure being applied orvacuum being drawn can be regulated by the signal processing unit suchthat the sample stops flowing through the analyte detection membranesystem for a period of time. As discussed herein, this can be referredto as a “dwell.” The dwell, for example, can be regulated by regulating(e.g. changing) the pressure that is applied to the analyte detectionmembrane system, which can be implemented or controlled by the signalprocessing unit. The dwell can also be regulated by regulating (e.g.changing) the vacuum that is being drawn through the analyte detectionmembrane system, which can be implemented or controlled by the signalprocessing unit. Any method of regulating the flow rate through theanalyte detection membrane system, including but not limited to, theflow rate through the conjugate pad and/or the test membrane can be usedand such method can be regulated or implemented by the signal processingunit.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the devices described herein andthroughout, comprises an analyte detection cartridge receptaclepositioning member. The detection cartridge receptacle positioningmember can be used, for example, to place the analyte detection membranesystem in the proper position to accept the sample and/or for the sampleto be analyzed. In some embodiments, the system is positioned forspectrometer analysis. The detection cartridge receptacle positioningmember can be, in some embodiments, motorized and/or controlled by thesignal processing unit. In some embodiments, the detection cartridgereceptacle positioning member is not motorized but can controlled by amanual controller, such as, but not limited to a lever or screw thatallows that receptacle's position to be modified. In some embodiments,the signal processing unit controls the movement of the analyte membranedetection receptacle by moving the analyte membrane detection receptaclemoving member. In some embodiments, the analyte detection cartridgereceptacle positioning member is in contact with analyte detectioncartridge receptacle.

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the devices described herein can comprisea waste receptacle. The waste receptacle can be in the interior of thedevice or outside but still contacting the device. The waste receptaclecan accept analyte detection membrane systems that have been used. Insome embodiments, as described herein, the analyte detection membranesystem is contained in a housing (e.g. cartridge). The housing can thenbe ejected into the waste receptacle. The ejection can be manual orautomated. In some embodiments, the ejection is controlled by a signalprocessing unit. In some embodiments, the signal processing unitcontrols an ejector that ejects the analyte detection membrane systemfrom the analyte detection membrane system receptacle into the wastereceptacle. Like all of the devices described herein, in someembodiments, the device comprises an analyte detection membrane system,which can or cannot be encased in a housing (e.g. cartridge).

In some embodiments of a device that can be used to detect multipleanalytes with a single signal, the pressure actuator contacts theanalyte detection membrane system. In some embodiments, the pressureactuator is attached to the device at a point that allows movement ofthe pressure actuator. In some embodiments, the pressure actuator isattached at a pivot point that allows the pressure actuator to pivot ata single contact point.

In some embodiments, the devices described herein comprise a display. Insome embodiments, the display is an electronic display. In someembodiments, the signal processing unit receives an input from thespectrometer and displays information on the display unit. The displayunit can be display various information, including but not limited to,the presence and/or absence of one or more analytes, status, and thelike.

In some embodiments, the present invention comprises detecting themultiple analytes using a device comprising a signal processing unit ora device described herein. In some embodiments, the method comprisescontacting the device with a sample that contacts the analyte detectionmembrane system within the device. The sample then flows through theanalyte detection membrane system. In some embodiments, the methodcomprises detecting the presence or absence of the analyte. In someembodiments, the detecting comprises the optical reader detecting anoptical signal from the analyte membrane system, the optical readercommunicating the optical signal to the signal processing unit, thesignal processing unit analyzing the optical signal to determine thepresence or absence of the analyte; and the signal processing unitdisplaying a result on the display unit. The displayed result can bevisual and/or audible. The signal analyzed can be a signal in a spectrumchosen from infrared spectrum; near infrared spectrum; visible spectrum,x-ray spectrum, ultra-violet spectrum, gamma rays, or electromagneticspectrum. In some embodiments, the signal is in the near-infraredspectrum. In some embodiments, the method comprises ejecting the analytedetection membrane system into a waste receptacle. In some embodiments,the signal processing unit is a computer.

Referring to the drawings, in some embodiments, FIGS. 1 through 36depict embodiments of devices, components of such representativedevices, and various views of such embodied devices that can be used inthe methods and/or in conjunction with or without other devices and/orsystems described herein.

These devices described herein are non-limiting and any other device,including other vertical flow devices, can be used to detect multipleanalytes according to the methods described herein using the bridgingcomplexes that are created using the various labels and capturereagents.

FIG. 8 depicts a device that can be used to detect multiple analyteswith a single signal comprising a first housing member (10), a buffercontainer (15), a second housing member (20), a groove for the slidingbutton (25), a sliding button (30), an inlet opening (35), a collar(40), and a test membrane (45). FIG. 8 depicts a test membrane (45)comprising two capture reagents. The first (10) and second (20) housingmembers can also be referred to as the lower and upper housing members,respectively. In FIG. 1, the sample would be applied through the inletopening (35) and can be allowed to vertically flow through to the testmembrane (45). In FIG. 8, the groove (25) allows the sliding button tomove, which when attached to the locking member moves the locking memberand can, in some embodiments, move the conjugate pad and change theposition of the force member.

FIG. 9 depicts a device that can be used to detect multiple analyteswith a single signal comprising a first housing member (10), a secondhousing member (20), a groove for the sliding button (25), a slidingbutton (30), an inlet opening (35), a collar (40), a test membrane (45),a conjugate pad (50), a plurality of absorbent members (e.g. pads) (55),an attachment member (60), a locking member (65), and a force member(70). FIG. 9 depicts the conjugate pad (50), test membrane (45) andabsorbent pad (55) arranged substantially parallel to one another. Theforce member (70) when in contact with the absorbent member would beapplying pressure that is substantially perpendicular to the conjugatepad. As can be seen in FIG. 9, a sample that is contacted with thedevice through the inlet opening (35) would flow vertically through theconjugate pad (50) to the test membrane (45). Not explicitly shown inFIG. 9, but in some embodiments, a the permeable membrane is alsosubstantially parallel to the conjugate pad (50) and to the testmembrane (45), with a first surface of the permeable membrane contactinga surface of the conjugate pad (50) a second surface of the permeablemembrane contacting a surface of the test membrane (45).

FIG. 10 depicts a conjugate pad (50), a permeable membrane (75), a testmembrane (45), and a plurality of absorbent members (55) that can beused to detect multiple analytes with a single signal. FIG. 10 depictsthe components that can be used to detect multiple analytes with asingle signal being substantially parallel with one another. FIG. 10depicts the permeable membrane (75) comprising an opening. This openingcan be used to allow visualization and detection of the test membrane'sresults.

FIG. 11 depicts a device that can be used to detect multiple analyteswith a single signal comprising a first housing member (10), a buffercontainer (15), a second housing member (20), a sliding button (30), atest membrane (45), a conjugate pad (50), a permeable membrane (75), aplurality of absorbent members (e.g. pads) (55), an attachment member(60), a locking member (65), and a force member (70). FIG. 11 alsodepicts the force member (70) comprising a shaft (72) and a head (71)where the head (71) is wider than the shaft (72).

FIG. 12 depicts a partial view of a device that can be used to detectmultiple analytes with a single signal comprising a first housing member(10), a locking member (65), a sliding button (30), and force member(70). FIG. 12 depicts the locking member (65) in contact with the forcemember (70) such that the force member (70) is in a raised method. FIG.12 also depicts the movement of the locking member (65) and the slidingbutton (30) away from the force member (70) allowing the force member tochange positions. In some embodiments, the change in position is thatthe force member is lowered.

FIG. 13 depicts a side cut away view of a device that can be used todetect multiple analytes with a single signal comprising a first housingmember (10), a second housing member (20), a sliding button (30), alocking member (65), a collar (40), an O-ring (41), a force member (70),and a support for the force member (73). The support for the shaft canbe, for example, part of the first housing member (10) and is shadeddifferently for example purposes only. FIG. 13 depicts the button (30)in contact with the locking member (65) in such a way that movement ofthe button (30) will move the locking member (65). Movement of thelocking member (65) will take away the support from the force member(70), which would allow the force member (70) to change positions. FIG.13 also depicts the shaft (72) and the head (71) of the force member.The head (71) creates a lip where the locking member (65) can slideunder and support the force member (70).

FIG. 14 depicts a partial view of a device that can be used to detectmultiple analytes with a single signal comprising a first housing member(10), a second housing member (20), an inlet opening (35), a testmembrane (45), a conjugate pad (50), a plurality of absorbent members(55), an attachment member (60), a locking member (65), and a forcemember (70). FIG. 8 depicts the attachment member (60) attached to theconjugate pad (50) and the locking member (65). FIG. 14 also depicts theconjugate pad being compressed against the second housing member (20)and the perimeter of the inlet opening (35). FIG. 14 depicts the head ofthe force member (71) applying the pressure by contacting the pluralityof absorbent members (55). In FIG. 14, a sample can be applied to thedevice through the inlet opening (35) so that the sample contacts theconjugate pad (50) and because of the pressure the sample throughvertical flow contacts the test membrane (45).

FIG. 15A depicts a partial view of a device that can be used to detectmultiple analytes with a single signal comprising a first housing member(10), a second housing member (20), an inlet opening (35), a testmembrane (45), a conjugate pad (50), a plurality of absorbent members(55), an attachment member (60), a locking member (65), and a forcemember (70). FIG. 8 depicts the movement of the locking member (65),which is attached to the attachment member (60). The movement of theattachment member (60), which is attached to the conjugate pad (50)moves the conjugate pad. FIG. 15A depicts the test force member (70)changing positions and a lessening or elimination of the pressure and/orcompression of the test membrane (45). FIGS. 15C and 15D also depictsthe movement of the conjugate pad (50) away from the inlet opening (35)revealing the test membrane (45) for visualization and/or detection.

FIG. 16 depicts an attachment member (60) attached to a conjugate pad(50). FIG. 16 depicts notches (51) in the conjugate pad (50) aslocations for the attachment member (60) to attach to. The attachmentmember can also be attached through other means such as throughadhesives, staples, and other forms of attachment.

FIG. 17 depicts a partial view of device that can be used to detectmultiple analytes with a single signal comprising a second housingmember (20), a plurality of pads or membranes (80), wherein theplurality of pads comprises a test membrane, a permeable membrane, andone or more absorbent members, and retaining members (85) that canretain the plurality of pads or membranes (80). FIG. 10 depicts thestructures that when the conjugate pad is moved the plurality of padsremains in place. Any means or other structure can be used to keep theplurality of pads in place.

FIG. 18 depicts a representative device that can be used to detectmultiple analytes with a single signal comprising a first housing member(1002) that further comprises a housing inlet (1006), and a secondhousing member (1004). In some embodiments, the first and second housingmembers can be constructed as a single unit. The housing inlet allowsfor the introduction of a sample onto the components inside the housing.The housing inlet can be of sufficient size to handle an appropriateamount of volume of a solution that is added to the device. In someembodiments, the size of the opening created by the housing inlet issufficient to handle about 0.1 to about 3 ml, about 0.1 to about 2.5 ml,about 0.5 to about 2.0 ml, about 0.1 to about 1.0 ml, about 0.5 to about1.5 ml, about 0.5 to about 1.0 ml, and about 1.0 to about 2.0 ml. Insome embodiments, the dimensions of the device are such that anydimension (e.g., width, depth, or height) is less than or equal to about5.08 cm (2.000 inches). In some embodiments, the height of the device isless than about 0.635 cm (0.250 inches), less than about 0.254 cm (0.100inches), less than about 0.191 cm (0.075 inches), less than about 0.165cm (0.065 inches), less than about 0.152 cm (0.06 inches), or less thanabout 0.140 cm (0.055 inches). In some embodiments, the height of thedevice is about 0.127 cm (0.050 inches). In some embodiments, the widthor depth of the device is less than or equal to about 5.08 cm (2.000inches), about 4.83 cm (1.900 inches), about 4.699 cm (1.850 inches),about 4.572 cm (1.800 inches), about 4.445 cm (1.750 inches), about4.191 cm (1.650 inches), about 4.064 cm (1.600 inches), or about 3.81 cm(1.500 inches). In some embodiments, the device is about 0.127 cm (0.050inches) in height, about 4.445 cm (1.750 inches) in depth, and about3.81 cm (1.500 inches) in width.

In some embodiments, the device that can be used to detect multipleanalytes with a single signal comprises a plurality of componentscomprising one or more of: a removable member, a conjugate pad, anadhesive member, a test membrane, an absorbent member, a force member, asupport member, or any combination thereof.

In some embodiments, the device that can be used to detect multipleanalytes with a single signal comprises a force member, a removablemember, a conjugate pad, a test membrane, an adhesive member and/or anabsorbent member. In some embodiments, the device comprises an analytedetection membrane system. In some embodiments, the analyte detectionmembrane system comprises a conjugate pad, a test membrane, and anabsorbent member. In some embodiments, the analyte detection membranesystem comprises an additional permeable membrane, but the device canalso be free of a permeable membrane. In some embodiments, the analytedetection membrane system comprises in the following order: a conjugatepad, an adhesive member, a test membrane, and an absorbent member.

FIG. 19 depicts an exploded view of the inside of a representativedevice that can be used to detect multiple analytes with a single signalcomprising a removable member (1005), a conjugate pad (1050), anadhesive member (1010), a test membrane (1030), an absorbent member(1040), and a support member (1020), wherein the support member furthercomprises an optional support member inlet (1025). The removable memberand the adhesive member can also comprise optional removable memberinlet (1008) and adhesive member inlet (1012), respectively. Suchcomponents could reside within, for example, the device of FIG. 18.

FIG. 20 depicts representative components of another representativedevice that can be used to detect multiple analytes with a single signalcomprising an adhesive member (1010), a support member (1020), a testmembrane (1030), and an absorbent member (1040). As can be seen in FIG.20, a sample can flow through the adhesive member (1010) and contact thetest membrane (1030).

FIG. 21 depicts an adhesive member (1010), a support member (1020), atest membrane (1030), and an absorbent member (1040). FIG. 21 depictsthe components being substantially parallel with one another. FIG. 21further depicts the support member (1020) comprising a support memberinlet (1025). This inlet can be used to allow the sample to verticallyflow through the device.

FIG. 22 depicts, in part, a conjugate pad (1050), a test membrane(1030), and an absorbent member (1040). FIG. 22 also depicts theconjugate pad in contact and/or attached to a removable member (1005).FIG. 22 also depicts the removable member being removed or moved awayfrom the device that can be used to detect multiple analytes with asingle signal, which also removes or moves away from the device theconjugate pad. The movement of the conjugate pad allows the testmembrane to be visualized, which facilitates analysis and detection ofanalytes, including multiple analytes with a single signal.

FIG. 23 depicts examples of force members (e.g. clips). Representativeforce members can come in a variety of shapes, sizes, andconfigurations, but each member applies pressure on the components thatare placed in or on the force member. Each force member can alsocomprise an opening (+) into which the analyze sample is applied. FIG.23 depicts non-limiting examples of force members with a first member(110) and a second member (100).

FIGS. 24A, 24B, 24C, and 24D depict, in part, a force member comprisinga first member (110), b) a second member (100), an inlet (115), and ananalyte detection membrane system (120). FIGS. 24A and 24B also depict,in part, a conjugate pad (1050). The conjugate pad is not seen in FIGS.24C and 24D. FIGS. 24C and 24D also depict, in part, a test membrane(1030) that is part of the analyte detection membrane system. FIG. 24Dalso depicts in part, a test membrane (1030) that has been reacted witha control, which is visualized by the band.

FIG. 25 depicts, in part, a container comprising a removable or movabletab (200), an inlet (210), a conjugate pad (1050), and the tab of theconjugate pad (1050). The tab of the conjugate pad (255) can be used toremove the conjugate pad (1050) from the device to expose the testmembrane. For example, a user could pull the tab of the conjugate pad(255) to remove the conjugate pad (1050) from the container. What is notvisualized is the analyte detection membrane system that is compressedbetween a first member (110) and a second member (100) as describedherein.

FIG. 26 depicts, in part, a first outer member (310), a second outermember (320), a movable or removable tab (330), and a conjugate pad(1050). The movable or removable tab (330) comprises an inlet thatexposes the conjugate pad (1050) so that the sample can be applied tothe conjugate pad. FIG. 26 does not show the first inner member (110)and the second inner member (100) compressing the analyte detectionmembrane system (120). The removable or movable tab (330) when moved orremoved, moves or removes the conjugate pad (1050), which allows thetest membrane to visualized and analyzed.

The removable member inlet within the removable member allows theintroduction of a sample onto the conjugate pad. The inlet can be ofsufficient size to handle an appropriate amount of volume of a solutionthat is added to the device. In some embodiments, the size of the inletis large enough to handle about 0.1 to about 3 ml, about 0.1 to about2.5 ml, about 0.5 to about 2.0 ml, about 0.1 to about 1.0 ml, about 0.5to about 1.5 ml, about 0.5 to about 1.0 ml, and about 1.0 to about 2.0ml. The removable member can also be constructed such that a portion ofthe removable member is permeable to solutions (i.e., the area definedby the removable member inlet) and another area is impermeable. Thepermeable area can act as an inlet because it would allow solutions tocross the removable member and contact the conjugate pad. The removablemember inlet can have any one of numerous shapes and sizes. In someembodiments, the first housing member serves as the removable member. Inother embodiments, the first housing member and the removable member areseparate components. In embodiments where the first housing member andthe removable member are separate components, at least a portion of thehousing inlet and removable member inlet overlap such that a solutioncan enter through both inlets.

In some embodiments, the removable member contacts a first surface of aconjugate pad. The removable member can also be attached to theconjugate pad. The removable member can be attached to the conjugate padby any means such that when the removable member is removed from thedevice or its position is changed, the conjugate pad is also removed orthe position of the conjugate pad is also changed. The removable membercan be attached to the conjugate pad with, for example, but not limitedto, an adhesive. Adhesives include, but are not limited to, glue, tape,or other substance that would allow the removable member and theconjugate pad to be attached to one another.

The removable member, in some embodiments, directly contacts theconjugate pad or indirectly contacts the conjugate pad through anotherlayer. The sample can be, in some embodiments, directly applied to theconjugate pad through the opening in the removable member.

FIG. 27A depicts, in part, an overhead view of a device that can be usedto detect multiple analytes with a single signal comprising a pluralityof portals (2036), an inlet (2035), and a housing member (2010). FIG.27A also depicts, in part, a portion of the channel system (2300) thatis visible through the portal (2301). FIG. 27B depicts, in part, anenlarged area of the device, specifically, the portal (2036). In theportal one can also see a plurality of capillary tubes (2301).

FIG. 28 depicts an underneath view of a device that can be used todetect multiple analytes with a single signal comprising a plurality offorce actuator outlets (2200), a housing member (2020), and a movingmember (2100).

FIG. 29 depicts, in part, a first housing member (2010), a secondhousing member (2020) a plurality of portals (2036), an inlet (2035), achannel system (2300), a plurality of capillary tubes (2301), aconjugate pad (2050), a plurality of test membranes (2045), and movablelocking member (2065). The channel system depicted in FIG. 29 isdepicted as consisting 3 branches, which is equal to the number ofanalyte detection membrane systems present in the device.

FIG. 30 depicts, in part, a second housing member (2020), a channelsystem (2300), a plurality of capillary tubes (2301), a conjugate pad(2050), a test membrane (2045), and an absorbent membrane (2055), and amovable locking member (2065), a flexible attachment member (2060), ananalyte detection membrane system (2400)

FIG. 31A depicts, in part, a plurality of force actuator outlets (2200),a channel system (2300), a plurality of capillary tubes (2301), aplurality of force members (2070), a movable locking member (2065), aplurality of movable locking member extensions (2068), a conjugate pad(2050), a plurality of flexible or non-flexible attachment memberextensions (2066) and nodule (2067), a test membrane (2045), andabsorbent membrane (2055).

FIG. 31B depicts, in part, a similar portion of the device shown in FIG.24A, however, the movable locking member (2065) has been rotated arounda central axis and the movable locking member extension (2068) no longersupports the force member (2070) and the force member has receded ordropped into the force actuator outlet (2200).

FIG. 32 depicts, in part, an exploded view of a device that can be usedto detect multiple analytes with a single signal comprising a channelsystem (2300), a conjugate pad (2050), a test membrane (2045), aplurality of force members (2070), a movable member (2100) that can turnthe movable locking member depicted (2065). FIG. 32 also depicts, inpart, movable locking member extension (2068), a plurality of flexibleor non-flexible attachment member extensions (2066) and nodule (2067), aflexible attachment member (2060), an outlet (2105), a second housingmember (2020), a plurality of force actuator outlets (2200), and aportion of an analyte detection membrane system (2047). The areacomprising the portion of the analyte detection membrane system (2047)has been enlarged and depicts, in part, a force member (2070), a testmembrane (2045), an absorbent member (2055), and portion of the movablelocking member extension (2068).

FIG. 33 depicts, in part, a housing (2020), a capillary channel (2301)and the channel system (2300). A portion of FIG. 33 has been enlarged todepict the conjugate pad (2050), the absorbent member (2055), and aplurality of capillary tubes (2301).

FIG. 34 depicts, in part, a cross-sectional view of a device that can beused to detect multiple analytes with a single signal comprising aplurality of portals (2036), an inlet (2035), a movable locking member(2065), a movable member that can move the movable locking member(2100), a force member (2700), a force actuator outlet (2200), aplurality of absorbent members (2055), a test membrane (2045), and amovable locking member extension (2068). FIG. 34 also depicts anexploded view of a portion of the analyte detection membrane systemcomprising a conjugate pad (2050), a permeable membrane (2056), and anabsorbent member (2055).

FIG. 35 depicts, in part, a non-limiting example of a movable lockingmember (2065) and a movable locking member extension (2068).

FIG. 36 depicts, in part, an exterior view and an interior view of ahousing comprising a plurality of portals (2036) and an inlet (2035).

FIG. 37 depicts, in part, an interior view and an exterior view of ahousing comprising a plurality of force actuator outlets (2200) and amovable member outlet (2105).

FIG. 38 depicts, in part, a device comprising a cartridge (3100) thatcan encompass an analyte detection membrane system, a force actuator(3200) and force release (3000), and outlet (3400), and an analytedetection membrane system receptacle (3300).

FIG. 39 depicts, in part, an enlarged view of the outlet (3400), thereceptacle (3300), and the cartridge (3100) depicted in FIG. 31.

FIG. 40 depicts, in part, an exploded view of a cartridge (3100)comprising a first housing member (3110), an inlet (3135), a conjugatepad (3350), a second housing member (3120), and a plurality of amembrane holders (3122).

FIG. 41 depicts, in part, a device for detecting an analyte comprisingan inlet (3335), a membrane system receptacle (3300), and display(3500).

FIG. 42 depicts, in part, the interior of the device that can be used todetect multiple analytes with a single signal depicted in FIG. 41. Thedevice comprises a cartridge comprising an analyte detection membranesystem (3100), a membrane system receptacle (3300), a force actuator(3200), a spectrometer (e.g. optical reader or photodetector (3600), anoptional conjugate pad remover (3201), an optional waste receptacle(3606), a motor and membrane system receptacle mover (3605/3607).

FIG. 43, shows the interior of a device that can be used to detectmultiple analytes with a single signal depicted in FIGS. 41 and 42 atvarious stages of use with the same components depicted in FIG. 35. FIG.43A depict the cartridge being inserted into the receptacle. FIG. 43Bdepicts the receptacle holding the cartridge being moved beneath theinlet for sample application and FIG. 43C depicts the sample beinganalyzed by the spectrometer.

FIG. 44 depicts an exploded view of a device that can be used for thedetection of a plurality of analytes with a single signal comprising afirst housing member (10), a second housing member (20), a groove forthe sliding button (25), a sliding button (30), an inlet opening (35), atest membrane (45), a conjugate pad (50), an additional membrane (51),an adhesive (52), a plurality of absorbent members (e.g. pads) (55), anattachment member (60), a locking member (65), and a force member (70).The components can be assembled as described and/or shown herein to makea device that can detect analytes using vertical flow.

FIG. 45 depicts a partially exploded view of a device that can be usedfor the detection of a plurality of analytes with a single signalcomprising a first housing member (10), a second housing member (20), agroove for the sliding button (25), a sliding button (30), an inletopening (35), a test membrane not seen, a conjugate pad (50), aplurality of absorbent members (e.g. pads) (not shown), an attachmentmember (60), a locking member (not shown), and a force member (notshown). Other variations of this device can also be made and used inaccordance with the methods described herein.

The embodiments are now described with reference to the followingexamples. These examples are provided for the purpose of illustrationonly and the embodiments should in no way be construed as being limitedto these examples, but rather should be construed to encompass any andall variations which become evident as a result of the teaching providedherein. Those of skill in the art will readily recognize a variety ofnon-critical parameters that could be changed or modified to yieldessentially similar results.

EXAMPLES Example 1

Two separate PCR reactions were performed with Shiga toxin genes as thetemplate that generated amplicons labeled with: 1) digoxigenin andbiotin, and 2) FITC and biotin. The amplicons were then either mixedtogether in the presence or absence of streptavidin (bridge unit) or ranseparately in a rapid flow through assay: Sample A) amplicon 1 alone,Sample B) amplicon 2 alone, or Sample C) amplicon 1+amplicon 2 with andwithout streptavidin. The flow through assay consisted of a solidsupport (nitrocellulose membrane) coated with anti-digoxigenin (firstcapture reagent) and colloidal gold particles coated with anti-FITCantibody. In this context, only Sample C with streptavidin generated asingle positive test signal whereas Sample A and Sample B, or Sample Cwithout streptavidin, resulted in a negative test.

Example 2 Detecting Multiple Analytes Using Amplicon Bridging

Materials:

PCR reagents: OneTaq Hot Start Polymerase (New England Biolabs); 5×Standard Reaction buffer; Haptenated MHALT1.RV (Integrated DNATechnologies (IDT)); Haptenated MgC.CH1AS (IDT); INV018.7E4 V_(H) genetemplate (ZG); dNTPs; dH₂O.

PCR was performed in a standard theremocycler at a ramp rate of 3-4°C./s. PCR reactions were run through a vertical flow assay such as thosedescribed herein including the Veriflow Cassette (Invisible Sentinel).

Amplicons were generated according to standard protocols. One ampliconwas generated dual labeled with fluorescein isothiocyanate (FITC) andtetramethylrhodamine (TAMRA) and another amplicon was generated that isdual labeled with TAMRA and digoxigenin (DIG). The DNA amplicons fromthe PCR reaction can be optionally precipitated. If precipitation wasperformed, it was done by either by EtOH or Isopropanol+1/10 v SodiumAcetate 3M, pH 5.2 precipitation. To facilitate precipitation, 1 uL oftRNA glycogen can also be added. The precipitation was allowed to takeplace at −20° C. for a minimum of 2 hours or −80° C. for 15 mins.Precipitated DNA was centrifuged at top speed for about 15 minutes.Supernatant was discarded the DNA pellet was allowed to air dry 15 mins.An optional second rinse with 20 uL ICE cold 70% EtOH can be performedfollowed by centrifugation and drying. DNA pellet was suspended with TE(Tris-HCl/EDTA) and the DNA was allowed to rehydrate for about 24 hoursat room temperature. The amplicons generated were generic sequences andnot specific to any particular bacteria.

The amplicons were mixed with a biotinylated antibody recognizing FITCand an antibody that recognized rhodamine (i.e. the TAMRA label). Themixture can be incubated for longer period of times, e.g. 5, 10, 15, 20,25, or 30 min, but such longer times were not necessary. The incubatedmixture was added to a Veriflow Cassette (vertical flow device), whichcontained a test membrane comprising an unlabeled anti-digoxigeninantibody and a conjugate pad containing streptavidin-gold conjugate. Thedevice detected the presence of the bridged complex, which contains bothamplicons with a single signal (the colloidal gold). The appropriatecontrols were performed and the colloidal gold was only detected whenall components necessary to create the bridging complex were present.Without wishing to be bound to any particular theory FIG. 3 illustratesthe complex that can be formed with the different components. Whenbridging complex is formed (see, FIG. 3) the colloidal gold signal isdetected. Other types of detectable signals can also be used. If one ofthe amplicons is not present no signal was detected. After the sample isrun through the device the streptavidin-colloidal gold complex isreleased from the conjugate pad and the conjugate pad is removed.Examples of how to make and use the vertical flow devices can be foundherein and in U.S. Pat. Nos. 8,012,770, 8,183,059 and U.S. patentapplication Ser. Nos. 13/500,997, 13/360,528, 13/445,233, each of whichis hereby incorporated by reference in its entirety. These resultsdemonstrate that two analytes can be specifically detected with a singledetectable signal, which in this example was colloidal gold. Thedetection of the signal was not dependent upon precipitating theamplicons after performing the PCR reaction step.

The disclosures of each and every patent, patent application,publication, and accession number cited herein are hereby incorporatedherein by reference in their entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. A method of concurrently detecting a firstanalyte of interest and a second analyte of interest comprising:contacting a solid support with a first analyte of interest, a secondanalyte of interest, a bridge unit comprising a second capture reagent,and a signal detection unit comprising a third capture reagent; anddetecting the presence or absence of the signal detection unit whichindicates the presence or absence of the first analyte of interest andsecond analyte of interest concurrently, wherein: a first capturereagent is affixed to the solid support; the first analyte of interestcomprises a first interaction unit that binds to the first capturereagent and a second interaction unit that binds to the bridge unit; andthe second analyte of interest comprises a first interaction unit and asecond interaction unit, wherein the first interaction unit binds thebridge unit; a signal detection unit that binds to: i) the secondanalyte, ii) to the second analyte's first interaction unit or secondinteraction unit, iii) to a component of the first and second analytecomplex, or iv)a component of an analyte-bridge complex that is onlypresent when the complex contains the first and second analytes.
 2. Themethod of claim 1, wherein the first and second interaction unit of thefirst analyte of interest and the first and second interaction unit ofthe second analyte of interest are each, independently, a heterologousinteraction unit.
 3. The method of claim 2, wherein the secondinteraction unit of the first analyte of interest and the firstinteraction unit of the second analyte of interest comprise the sameheterologous interaction unit.
 4. The method of claim 2, wherein thefirst interaction unit of the first analyte of interest and the secondinteraction unit of the second analyte of interest comprise the sameheterologous interaction unit.
 5. The method of claim 1, wherein thefirst and second analytes of interest are, independently, anamplification product, a peptide, a sugar, an antigen, a nucleic acidmolecule, or any combination thereof.
 6. The method of claim 1, whereinthe signal detection unit comprises a radioactive tag, colloidal gold, afluorescent tag, a nanoparticle, an emissive nanoparticle, a quantumdot, a magnetic particle, or an enzyme.
 7. A method of concurrentlydetecting a first analyte of interest, a second analyte of interest, anda third analyte of interest with a single signal comprising: contactingthe first, second, and third analytes of interest with a solid support,a first bridge unit, a second bridge unit, and a signal detection unit;and detecting the presence of the signal detection unit which indicatesthe presence of the first, second, and third analytes of interestconcurrently with a single signal, wherein: the first analyte ofinterest comprises a first interaction unit and a second interactionunit; the second analyte of interest comprises a first interaction unitand a second interaction unit; the third analyte of interest comprises afirst interaction unit and a fifth interaction unit; the solid supportcomprises a first capture reagent that binds to the first interactionunit of the first analyte of interest; the first bridge unit binds tothe second interaction unit of the first analyte of interest and thefirst interaction unit of the second analyte of interest; the secondbridge unit binds to the second interaction unit of the second analyteof interest and the first interaction unit of the third analyte ofinterest; and the signal detection unit binds to the second interactionunit of the third analyte of interest.
 8. The method of claim 7, whereinthe first bridge unit is a multivalent capture reagent.
 9. The method ofclaim 8, wherein the multivalent capture reagent is an immunoglobulin.10. A method of concurrently detecting a plurality of analytes with asingle signal, the method comprising: i) contacting a device fordetecting a plurality of analytes with a single signal with one or moresamples comprising a plurality of analytes, wherein the devicecomprises: a housing comprising: an inlet opening in fluid contact witha conjugate pad; a force member; a slidable locking member contactingthe force member; an attachment member contacting the force member; asliding button contacting the attachment member; and a detectionmembrane system comprising the conjugate pad, a test membrane, and anabsorbent member, at least a portion of the conjugate pad, testmembrane, and absorbent member are substantially parallel to each other,the force member contacts the detection membrane system and is capableof applying pressure substantially perpendicular to the detectionmembrane system, the sliding button moves the slidable locking member,the conjugate pad comprises a signal detection unit comprising a thirdcapture reagent; the test membrane comprises a first capture reagentaffixed to the test membrane; wherein the one or more samples comprisesa first analyte of interest, a second analyte of interest, and a bridgeunit comprising a second capture reagent, wherein the first analyte ofinterest comprises a first interaction unit that binds to the firstcapture reagent and a second interaction unit that binds to the bridgeunit, and the second analyte of interest comprises a first interactionunit that binds the bridge unit and a second interaction unit; whereinthe signal detection unit comprising the third capture reagent binds tothe second analyte, to the second analyte's first interaction unit orsecond interaction unit, to a component of the first and second analytecomplex, or to a component of the bridge unit that that is only presentwhen the complex contains the first and second analytes; and ii)detecting the presence or absence of the signal detection unit whichindicates the presence or absence of the first analyte of interest andsecond analyte of interest concurrently.
 11. The method of claim 10,wherein detecting comprises moving the conjugate pad after a portion ofthe one or more samples has contacted and flowed through the conjugatepad, thereby exposing at least a portion of the test membrane fordetection of the signal detection unit to indicate the presence orabsence of the plurality of analytes with a single signal.
 12. Themethod of claim 10, wherein the first and second analyte are amplicons.13. The method of claim 10, wherein the first and second analytes arePCR reaction products.
 14. The method of claim 10, wherein: the firstanalyte's first interaction unit is a digoxigenin label, the firstanalyte's second interaction unit is a rhodamine label, second analyte'sfirst interaction unit is a rhodamine label, or second analyte's secondinteraction unit is a fluorescein label.
 15. The method of claim 10,wherein the third capture reagent binds to the second analyte's secondinteraction unit.
 16. The method of claim 10, wherein the third capturereagent is a biotinylated capture reagent.
 17. The method of claim 10,wherein the signal interaction unit is coated with streptavidin.
 18. Themethod claim 10, wherein the signal interaction unit is streptavidincoated colloidal gold.
 19. The method of claim 10, wherein the first andsecond analytes are nucleic acid amplification products, wherein: thefirst analyte comprises a digoxigenin label and a rhodamine label; thesecond analyte comprises a rhodamine label and a fluorescein label; thefirst capture reagent is an anti-digoxigenin label antibody; the secondcapture reagent is an anti-rhodamine label antibody; the third capturereagent is a biotinylated anti-fluorescein label antibody; and thesignal interaction unit is streptavidin coated colloidal gold.